the roles of presenilin and fkbp14 in drosophila ... · iv acknowledgements i would like to thank...

THE ROLES OF PRESENILIN AND FKBP14 IN DROSOPHILA DEVELOPMENT

AND NOTCH SIGNALLING

by

Diana L. van de Hoef

A thesis submitted in conformity with the requirements

for the degree of Doctorate of Philosophy

Department of Molecular Genetics

University of Toronto

© Copyright by Diana L. van de Hoef 2008

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THESIS TITLE: The Roles of Presenilin and FKBP14 in Drosophila Development and

Notch Signalling

AUTHOR: Diana L. van de Hoef

DEGREE: Doctorate of Philosophy

DEPARTMENT: Department of Molecular Genetics

UNIVERSITY: University of Toronto

YEAR: 2008

ABSTRACT

The multimolecular g-secretase complex cleaves type 1 transmembrane proteins such as

Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex

comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2,

nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of

g-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease.

To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier

screen to identify Psn- interacting genes. One of the genes that was identified, known as

FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the

ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs

and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin

enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx

defective 1 and Psn in the ER, suggesting a role in γ-secretase stability. Consistent with this,

I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in

vivo.

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To further characterize the role of FKBP14 in development, I analyzed its expression

pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to

embryonic hemocytes and larval tissues, in addition to being expressed in developing egg

chambers. FKBP14 function is required during development, since FKBP14 null mutants are

recessive lethal. These mutants exhibited defects in larval disc development that resulted in

eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent

phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues,

suggesting a possible involvement in apoptosis regulation. I then examined the effects of

FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly,

co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a

role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14

mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data

demonstrate an essential role for FKBP14 during development, particularly in Psn protein

maintenance and Notch signalling.

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ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr. Gabrielle Boulianne, for her support, mentorship and

encouragement throughout my degree. This thesis was possible because of the confidence and skills

that I learned under her guidance, and for that I am particularly grateful.

I am also grateful to my committee members, Dr. Johanna Rommens for invaluable advice

and mentorship, and Dr. Craig Smibert for helpful insight, encouragement and support. Their

guidance and inspiration through the years have motivated me, and have helped shape my ideas about

research.

I thank my colleagues, Jamie Hughes and Izhar Livne-Bar, for assistance with the genetic

modifier screen. Additionally, I thank Kinga Michno for her contributions to Chapter 2.

I met many colleagues throughout my stay in the Boulianne lab, both past and present, and I

gratefully acknowledge Cosimo Commisso, Maia Renihan, Jamie Hughes, Sabrina Kim, Brenda

Chow, Peter Leventis, Kinga Michno, David Knight, Julia Maeve Bonner, Yuanfang Liu, Patricia

Zeemann, Oxana Gluscencova, Michael Garroni, Philip Harvey, Lara Skwarek, Natali Iliadi, Kostas

Iliadi, Edward Yeh, and Hao Xu for their advice and support.

Personally, I am grateful to Jamie Hughes and Maia Renihan for their support. I am

especially grateful to Cosimo Commisso for his unwavering support, advice and encouragement.

Finally, I thank my family, especially my parents John and Arlene van de Hoef, for their constant

encouragement and support.

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Table of Contents

CHAPTER 1: INTRODUCTION.................................................................................................1

1.1 Summary...........................................................................................................................2

1.2 Overview of Alzheimer’s disease .......................................................................................2

1.2.1 The role of Presenilin in Alzheimer’s disease .................................................................3

1.2.2 Overview of the γ-secretase complex.............................................................................4

1.2.3 Assembly and regulation of γ-secretase..........................................................................6

1.2.4 The spatial paradox ......................................................................................................7

1.3 Targets of the γ-secretase ..................................................................................................8

1.3.1 The role of Presenilin-dependent γ-secretase activity in Notch signalling .........................8

1.3.2 Summary of the Notch signalling pathway .....................................................................9

1.3.3 Involvement of Notch signalling in lateral inhibition..................................................... 11

1.3.4 Notch activity in other signalling paradigms................................................................. 11

1.3.5 Notch activity during hematopoiesis ............................................................................ 15

1.4 Presenilin function independent of its γ-secretase activities.............................................16

1.4.1 Intracellular calcium homeostasis, regulated by presenilin, is defective in AD................ 17

1.4.2 The role of presenilin in the apoptotic signalling pathway ............................................. 18

1.5 Overview of FKBPs.........................................................................................................20

1.5.1 FKBP function regulates intracellular calcium signalling .............................................. 20

1.5.2 The role of FKBPs during the unfolded protein response............................................... 22

1.5.3 FKBP14 localizes to the endoplasmic reticulum to facilitate protein folding................... 22

1.6 Effects of the immunosuppressant, FK506, on FKBP function ........................................23

1.6.1 FK506 immunosuppression elicits FKBP12 gain-of-function during T-cell inhibition..... 24

1.6.2 FK506 treatment epidemiology ................................................................................... 25

1.7 Rationale .........................................................................................................................26

CHAPTER 2: A GENETIC SCREEN TO IDENTIFY GENES THAT INTERACT WITH

PRESENILIN IN DROSOPHILA MELANOGASTER ...............................................................27

2.1 Summary.........................................................................................................................28

2.2 Introduction ....................................................................................................................28

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2.3 Materials and Methods....................................................................................................31

2.3.1 Drosophila genetics.................................................................................................... 31

2.3.2 Drosophila stocks....................................................................................................... 32

2.3.3 Immunoblot Analysis ................................................................................................. 32

2.4 Results.............................................................................................................................33

2.4.1 Presenilin overexpression causes Notch-related phenotypes in the notum and wing ........ 33

2.4.2 A Screen to identify Presenilin modifiers..................................................................... 33

2.4.2.1 Calcium signalling components ............................................................................ 35

2.4.2.2 Regulators of stress response and protein folding .................................................. 36

2.4.2.3 Cell cycle and apoptotic factors............................................................................ 36

2.4.2.4 Signal transduction and trafficking components..................................................... 43

2.4.2.5 Hematopoietic factors.......................................................................................... 44

2.4.2.6 Alzheimer’s disease-associated factors.................................................................. 44

2.4.3 Confirmation of the genetic interactions between Presenilin and components of

intracellular calcium signalling............................................................................................ 45

2.4.4 Psn overexpression levels are reduced in FKBP14 mutants ........................................... 45

2.5 Discussion........................................................................................................................47

2.6 Acknowledgements..........................................................................................................53

CHAPTER 3: THE INTERACTION BETWEEN FKBP14 AND PRESENILIN MEDIATES

NOTCH SIGNALLING DURING DROSOPHILA DEVELOPMENT.........................................54

3.1 Summary.........................................................................................................................55

3.2 Introduction ....................................................................................................................55



3.3.2 Drosophila stocks....................................................................................................... 57

3.3.3 RT-PCR..................................................................................................................... 57

3.3.4 Immunoblot Analysis and Immunoprecipitation ........................................................... 58

3.3.5 Immunohistochemistry ............................................................................................... 58

3.3.6 Microscopy................................................................................................................ 59

3.3.7 Cell culture ................................................................................................................ 59

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3.4 Results.............................................................................................................................59

3.4.1 Molecular Characterization of Drosophila FKBP14...................................................... 59

3.4.2 Drosophila FKBP14 is an ER-resident protein ............................................................. 62

3.4.3 FKBP14 mutants exhibit defects in sense organ development........................................ 62

3.4.4 Loss of FKBP14 function impairs Notch signalling at the wing margin and in proneural

clusters .............................................................................................................................. 65

3.4.5 FKBP14 is not required for Notch trafficking to the plasma membrane .......................... 71

3.4.6 FKBP14 mutants genetically interact with Notch and Psn ............................................. 74

3.4.7 FKBP14 colocalizes and binds with Psn in the ER........................................................ 76

3.4.8 FKBP14 function maintains Psn protein levels ............................................................. 77

3.5 Discussion........................................................................................................................78


CHAPTER 4: ANALYSIS OF FKBP14 FUNCTION DURING DEVELOPMENT.....................82

4.1 Summary.........................................................................................................................83

4.2 Introduction ....................................................................................................................83


4.3.1 Plasmids .................................................................................................................... 84


4.3.3 Immunoblot Analysis ................................................................................................. 85

4.3.4 Immunostaining ......................................................................................................... 85

4.3.5 Cell culture ................................................................................................................ 86

4.4 Results.............................................................................................................................86

4.4.1 FKBP14 is detected in all stages of development.......................................................... 86

4.4.2 FKBP14 is broadly expressed in developing egg chambers and early-stage embryos, and is

specifically localized in late-stage embryonic hemoctyes....................................................... 88

4.4.3 FKBP14 is broadly expressed in third instar larval discs ............................................... 90

4.4.4 FKBP14 overexpression increases Psn protein levels and affects Notch signalling.......... 90

4.5 Discussion........................................................................................................................96


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CHAPTER 5: EFFECTS OF THE IMMUNOSUPPRESSANT DRUG FK506 ON THE GAMMA-

SECRETASE COMPLEX..........................................................................................................99

5.1 Summary.......................................................................................................................100

5.2 Introduction ..................................................................................................................100

5.2 Materials and Methods..................................................................................................101

5.2.1 Plasmids .................................................................................................................. 101

5.2.2 Immunoblot Analysis ............................................................................................... 102

5.2.3 Immunostaining ....................................................................................................... 102

5.2.4 Cell culture .............................................................................................................. 103

5.3 Results and Discussion ..................................................................................................103

5.3.1 FKBP14 and Aph-1 colocalize in the ER ................................................................... 103

5.3.2 FK506 decreases Aph-1, Pen-2 and Psn protein levels in S2 cells ................................ 104

5.4 Conclusions ...................................................................................................................105

5.5 Acknowledgements........................................................................................................106

CHAPTER 6: PROSPECTIVES AND FUTURE DIRECTIONS..............................................107

6.1 Summary.......................................................................................................................108

6.2 Involvement of Psn in numerous intracellular activities ................................................108

6.2.1 Examining the protein-protein interactions between Psn and the candidate genes.......... 108

6.2.2 Examining the effects of Psn FAD-linked mutations on Psn modifier genes ................. 109

6.2.3 Examining a role for Psn in Drosophila hematopoiesis ............................................... 109

6.2.4 Characterizing the candidate gene mutations .............................................................. 110

6.3 Examining the interaction between Psn and FKBP14....................................................110

6.3.1 Characterizing the site of interaction between Psn and FKBP14 .................................. 110

6.3.2 Analysis of a direct interaction between FKBP14 and Psn in the ER ............................ 111

6.3.3 Examining the interaction between Psn and FKBPs .................................................... 112

6.4 Examining FKBP14 function in vivo..............................................................................112

6.4.1 Determining FKBP14 function in oogenesis............................................................... 113

6.4.2 Determining the role of FKBP14 in hematopoiesis ..................................................... 113

6.4.3 Examining FKBP14 function in calcium homeostasis ................................................. 114

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6.4.4 Determining FKBP14 function in apoptosis ............................................................... 115

6.4.5 Is FKBP14 function involved in proneural gene activity?............................................ 115

6.5 Analyzing the effects of FK506 on the γ-secretase complex............................................116

6.5.1 Determining FKBP expression in S2 cells.................................................................. 116

6.5.2 Examining the effects of FK506 inhibition of FKBPs in cell culture on γ-secretase....... 117

6.6 Conclusions ...................................................................................................................118

REFERENCES...................................................................................................................119

x

List of Tables

Table 2.1 Molecular Classification of Presenilin Modifiers --------------------------------- 37

List of Figures

Figure 1.1 Schematic representation of the γ-secretase complex in vertebrates..................................5

Figure 1.2 Proteolytic activation of APP and the Notch receptor ......................................................6

Figure 1.3 Schematic of the Notch signalling pathway .................................................................. 10

Figure 1.4 Notch-dependent lateral inhibition specifies bristle sense organ development ................. 12

Figure 1.5 Notch activity specifies cell fates through the process of inductive signalling ................. 13

Figure 1.6 Schematic representation of the anterior portion of a Drosophila ovariole ...................... 14

Figure 1.7 Drosophila hematopoietic cell lineage ......................................................................... 16

Figure 1.8 Domain structure of 6 characterized human FKBPs ...................................................... 21

Figure 2.1 Suppression or enhancement of Psn-dependent Notch-related phenotypes in adult nota and

wings................................................................................................................................. 34

Figure 2.2 Cam and FKBP14 null mutants suppress Psn overexpression phenotypes ....................... 46

Figure 2.3 Psn protein levels are mildly reduced in FKBP14D58 heterozygotes ................................ 47

Figure 3.1 Drosophila FKBP14 shows homology to vertebrate FKBP14 ........................................ 60

Figure 3.2 FKBP sequence alignment........................................................................................... 61

Figure 3.3 Drosophila FKBP14 localizes to the ER....................................................................... 62

Figure 3.4 FKBP14 mutants exhibit reduced FKBP14 levels ......................................................... 63

Figure 3.5 FKBP14 mutants exhibit defects in larval CNS and disc development, and increased levels

of apoptosis in third instar larval CNS and discs ................................................................... 65

Figure 3.6 FKBP14 mutants display defects in eye development.................................................... 66

Figure 3.7 FKBP14 is required for Notch target gene expression at the presumptive wing margin.... 67

Figure 3.8 FKBP14 is required for Notch target gene expression in wing pouch and hinge regions... 69

Figure 3.9 FKBP14 is required for SOP development and Notch target gene expression in third instar

larval wing discs................................................................................................................. 70

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Figure 3.10 FKBP14 function is involved in proneural gene expression in third instar larval wing

discs .................................................................................................................................. 72

Figure 3.11 FKBP14 is required for bristle formation in adult nota................................................. 73

Figure 3.12 FKBP14 functions downstream of Notch trafficking to the plasma membrane .............. 74

Figure 3.13 Quantitation of the genetic interactions between FKBP14 and Notch............................ 75

Figure 3.14 FKBP14 genetically and physically interacts with Psn................................................. 76

Figure 3.15 Loss of FKBP14 affects Psn protein levels ................................................................. 77

Figure 4.1 FKBP14 protein expression is required during development .......................................... 87

Figure 4.2 FKBP14 expression is detected in follicle cells, nurse cells and oocytes in mid-stage egg

chambers............................................................................................................................ 88

Figure 4.3 FKBP14 localization in embryonic hemocytes and fat body........................................... 89

Figure 4.4 FKBP14 is broadly expressed in larval imaginal discs................................................... 91

Figure 4.5 FKBP14 overexpression increases Psn protein levels in S2 cells .................................... 92

Figure 4.6 FKBP14 and Psn proteins that express C-terminal tags exhibit similar subcellular

localization as endogenous proteins ..................................................................................... 93

Figure 4.7 FKBP14 overexpression increases Psn protein levels in vivo ......................................... 94

Figure 4.8 Quantitation of FKBP14 and Psn overexpression Notch-related bristle phenotypes ......... 95

Figure 5.1 FKBP14 partially colocalizes with Aph-1 in vivo........................................................ 104

Figure 5.2 FK506 treatment disrupts Aph-1 and Pen-2 overexpression levels, and endogenous PsnNTF

protein levels.................................................................................................................... 106

Figure 6.1 A multiple sequence alignment of FKBP14 orthologs from fly and yeast...................... 112

xii

Abbreviations

Aβ Amyloid-beta

Ac Achaete

AD Alzheimer’s disease

AICD APP intracellular domain

Aph-1 anterior pharynx defective 1 (fly)

APH1A anterior pharynx defective 1 homolog A (human)

APH1B anterior pharynx defective 1 homolog B (human)

APP Amyloid Precursor Protein

Cam Calmodulin

CNS Central Nervous System

CTF C-terminal fragment

Ct Cut

DGRC Drosophila Genome Resource Center

ER endoplasmic reticulum

E(spl) Enhancer of Split protein complex

FAD familial Alzheimer’s disease

FKBP FK506-binding protein

FKBP1A FK506-binding protein 1A (FKBP12)

FKBP2 FK506-binding protein 2 (FKBP13)





Gcm glial cells missing

Lz Lozenge

Nβ Notch-1 amyloid-beta- like peptide

NEXT Notch extracellular truncation

Nct nicastrin (fly)

NCSTN nicastrin (human)

xiii

NICD Notch intracellular domain

NTF N-terminal fragment

p3 APP 3-kDa derivative peptide

Pen-2 presenilin enhancer (fly)

PSENEN presenilin enhancer 2 homolog (human)

PPIase peptidyl-prolyl cis-trans isomerase

pnr pannier

Psn Presenilin (fly)

PSEN presenilin (human)

PSEN1 presenilin-1 (human)

PSEN2 presenilin-2 (human)

Rer1 retrieval to the endoplasmic reticulum 1

RyR Ryanodine receptor

Sens Senseless

Ser Serrate

SOP sense organ precursor

Su(H) Suppressor of Hairless

Srp Serpent

TACE TNF-alpha converting enzyme

Ush U-shaped

Vkg viking

Wg Wingless

WT wild type

1

CHAPTER 1

INTRODUCTION

2

1.1 Summary

Presenilin is an aspartyl protease that was initially identified as a causative factor in

Alzheimer’s disease (AD). Subsequently, presenilin was implicated in Notch signalling during

development, since presenilin mutants exhibit Notch-related defects. Presenilin functions as

part of a multimolecular protein complex referred to as γ-secretase that proteolytically cleaves

numerous type 1 transmembrane proteins, including Amyloid Precursor Protein (APP) and

Notch. APP cleavage leads to Aβ production, and defects in this process are involved in AD

pathology. Notch processing activates signalling that is required for numerous developmental

decisions.

The goal of my thesis was to characterize the role of presenilin in development and in

particular, within the Notch signalling pathway. To this end, I performed a genetic screen to

identify Presenilin- interacting factors in Drosophila. This led to the identification of numerous

factors, including an FK506-binding protein known as FK506-binding protein 14 (FKBP14),

which may be involved in protein folding.

In my introduction, I will first provide an overview of AD and discuss the molecular

composition of the γ-secretase complex as it is currently understood. I then describe γ-secretase

activities that have been implicated in the pathogenesis of AD and in the Notch signal

transduction pathway. I will include a summary of the roles of Notch signalling during

oogenesis and larval development. I also discuss recent advances in understanding Presenilin

function, particularly its roles in calcium homeostasis and apoptosis, and how FKBPs may be

implicated in these processes. Since FKBP14 is a member of a large family of FKBPs, I will

then describe FKBP function during development. Interestingly, FKBPs are targets for a

common immunosuppressant drug, known as FK506, and I will discuss the effects of FK506 on

FKBP function.

1.2 Overview of Alzheimer’s disease

Alzheimer’s disease is a progressive neurodegenerative disorder that is believed to affect

nearly 24 million people worldwide [1]. AD prevalence increases with age, affecting nearly

50% of people over the age of 85 [2]. Risk factors also include family history, genetics and

Down’s syndrome [3, 4]. Familial AD (FAD) patients inherit the disease and present symptoms

before the age of 65 [4]. These cases are referred to as early-onset, and account for 1-5% of the

3

total number of AD cases, which are generally late-onset and sporadic [4]. FAD pathology is

associated with mutations in the presenilin-1, presenilin-2 and Amyloid Precursor Protein (APP)

genes. APP is a type 1 transmembrane protein that traverses the plasma membrane, and

undergoes several proteolytic cleavage events. In the amyloidogenic pathway, its extracellular

domain is initially cleaved by β-secretase [5], and the remaining substrate is recognized and

cleaved by the presenilin-dependent γ-secretase complex, releasing extracellular Aβ peptides

[5]. Presenilins are multipass transmembrane proteins with cytosolic amino- and carboxy-

termini and a cytosolic hydrophilic loop between transmembranes 6 and 7 that is

endoproteolytically cleaved, generating an active Psn heterodimer within the γ-secretase

complex [6-8]. Mutations in vertebrate presenilins and APP affect Aβ peptide production,

releasing a longer version of the Aβ peptide that aggregates and forms extracellular neurotoxic

plaques which are believed to be a leading cause of AD pathology [9-14]. Another hallmark of

Alzheimer’s disease is the appearance of intracellular neurofibrillary tangles, composed of a

microtubule binding protein known as Tau, that is abnormally hyperphosphorylated, leading to

neuronal loss [15].

1.2.1 The role of Presenilin in Alzheimer’s disease

Presenilins (PSEN) were initially identified through genetic linkage studies in patients

with an aggressive early-onset form of AD [12, 16]. Over 100 mutations in vertebrate PSEN1

have been identified in Alzheimer’s pedigrees, and mutations in PSEN2 and APP have also been

identified [4, 17]. The majority of PSEN1 mutations are missense substitutions, primarily

within highly conserved transmembrane domains and in the cytoplasmic loop region, although

rare deletions or insertions have also been identified [4, 17]. Approximately 7 PSEN2 mutations

and 19 APP mutations have been identified in AD patients [4, 18]. Together, these mutations

account for up to 50% of known early-onset AD cases. The central paradigm of early-onset AD

pathology is that PSEN mutations disrupt APP cleavage, accelerating the production of Aβ42

peptides that aggregate extracellularly and show greater neurotoxicity than the normally

produced Aβ40 peptides [19], and consequently, loss of cognitive functions. In initial stages, it

is believed that Aβ42 peptides cause a moderate increase in levels of oxidative stress in neurons,

and as the disease progresses, more Aβ peptides accumulate, causing an increase in oxidative

stress and cytosolic calcium release, leading to neuronal degeneration and apoptosis [20]. This

model is derived from studies of the effects of Aβ42 in neuronal cell culture that show increased

4

intracellular calcium levels, enhanced membrane lipid peroxidation and free radical production

and increased levels of apoptosis [21, 22]. Oxidative stress may also play a role in Tau- induced

neurotoxicity, leading to apoptosis [23].

PSEN1 and PSEN2 mutations alter APP cleavage, and account for the majority of known

early-onset AD cases [4], thus there has been much interest in determining the roles of PSEN in

vivo. While the precise function of PSEN has yet to be fully determined, it is thought to

function as the catalytic component of the large proteolytic protein complex, γ-secretase, which

localizes to various intracellular locations, including ER, Golgi and endosomal compartments

[24-27]. In the following sections, I will describe the roles of presenilin in the γ-secretase

complex and its proposed functions during development and AD pathogenesis.

1.2.2 Overview of the γ-secretase complex

The current accepted model for the core g-secretase complex shows four essential proteins,

known as anterior pharynx defective 1 (Aph-1), presenilin enhancer (Pen-2), nicastrin (Nct) and

Presenilin (Psn) in flies [28] and APH1, PSENEN, NCSTN and PSEN in vertebrates (Fig. 1.1;

[13, 14, 29]). This complex activates the final intramembrane cleavage of type 1

transmembrane domain proteins, such as Notch and APP, liberating their intracellular domains

for translocation to the nucleus (Fig. 1.2; [30]). The precise stoichiometry of the complex has

been debated, however it has been shown that expression of all four components in yeast, which

lack endogenous γ-secretase activity, is sufficient to produce Aβ and AICD in vitro and thus,

can reconstitute biological activity of the complex [31]. At present, the precise role of each

component has yet to be defined, although numerous studies have suggested putative functions

which I will now review.

Presenilin is synthesized as a precursor protein of approximately 50 kDa within the ER

that is rapidly degraded unless incorporated into a larger complex [32, 33]. It is then cleaved by

an unknown enzyme within the cytoplasmic loop yielding a 28 kDa amino-terminal fragment

(PsnNTF) and a 17 kDa carboxy-terminal fragment (PsnCTF) that are both required for γ-secretase

cleavage of type 1 transmembrane domain proteins [32, 34]. Since mutations within conserved

aspartyl residues in presenilin abolish g-secretase activity [34-37], and aspartyl protease

inhibitors selectively cross-link to presenilin and inhibit g-secretase cleavage of substrates [38-

5

40], it has been postulated that presenilin functions as an aspartyl protease within the catalytic

core of the γ-secretase complex [41, 42].

Nicastrin is a type 1 integral membrane protein that displays both immature, and mature

glycosylated forms [43], although glycoslyation may not be essential to γ-secretase activity [44].

It was shown to physically associate with presenilins in human brains and mammalian cell-

based assays [45, 46]. Nicastrin is proposed to function as a scaffolding molecule, required for

trafficking of the complex and for substrate recognition, possibly at the cell surface [44, 47-49].

Aph-1 and Pen-2 were originally identified in a genetic screen in C. elegans [46]. Aph-1

has been implicated in complex stability involving intermolecular and intramolecular

interactions [50]. Drosophila Pen-2, or PSENEN in vertebrates, is incorporated late during

assembly of the complex, facilitating presenilin endoproteolysis [28, 51, 52]. After cleavage,

the presenilin heterodimer is stabilized through intermolecular interactions with the C-terminus

of Pen-2, yielding an active g-secretase complex [28, 51-53]. Whether these four proteins are

sufficient to carry out the more than 30 known γ-secretase substrate cleavages [8, 54], or if they

require additional proteins, remains to be determined.

Figure 1.1 Schematic representation of the γ-secretase complex in vertebrates. Presenilin enhancer 2 homolog (red; PSENEN) directly interacts with the N-terminal fragment of presenilin (PSEN; light blue). Presenilin is a multipass transmembrane protein with up to 9 membrane-spanning segments [55], cytosolic amino- and carboxy-termini and a cytosolic hydrophilic loop between transmembranes 6 and 7. The arrow indicates the endoproteolytic site that generates an active heterodimer and asterisks mark the position of critical aspartate residues. Anterior pharynx defective 1 homolog (APH1; green) and nicastrin (NCSTN; dark blue) interact directly, and bind to presenilin C-terminal fragments. Adapted from [6-8]. A similar composition of the γ-secretase complex has been identified in flies [31]. Drosophila encodes for a single Presenilin protein that gives rise to two isoforms [56], and single anterior pharynx defective 1 (Aph-1), nicastrin (Nct) and presenilin enhancer 2 (Pen-2) proteins [28, 57].

Cytosol APH1

* *

Lumen NTF CTF

NCSTN PSENEN

PSEN

6

Figure 1.2 Proteolytic activation of APP and the Notch receptor. Amyloidogenic processing of human APP (hAPP) involves an initial cleavage by β-secretase that releases soluble extracellular fragments (APPsβ) and a C99 stub. The γ-secretase heterogeneously cleaves C99 at the γ-, z-, and e-sites secreting Aβ-peptides and releasing AICD for translocation to the nucleus (Periz and Fortini). The non-amyloidogenic pathway of APP involves cleavage by a-secretase releasing neuroprotective soluble fragments (APPsa), and a C83 stub that is processed by γ-secretase releasing p3 peptides of unknown function and AICD [58, 59]. Similarly, in the canonical Notch signalling pathway, S1 cleavage of mouse Notch1 (mNotch1) by a furin peptidase in the trans-Golgi network generates a stable heterodimer that transports to the cell surface. Upon ligand binding (not shown), S2 cleavage by the metalloprotease Kuzbanian results in ectodomain shedding, an essential step prior to γ-secretase processing at S3 and S4 sites. These last two cleavage events mimic the e - and γ-site cleavages of APP, producing Nβ peptides, of unknown function, and the NICD that translocates to the nucleus [60]. Abbreviations: Aβ (amyloid-beta), AICD (APP intracellular domain), APP (Amyloid Precursor Protein), 3-kDa derivative peptide (p3), TNF-a converting enzyme (TACE), Notch extracellular truncation (NEXT), Notch-1 Aβ-like peptide (Nβ) and Notch intracellular domain (NICD). Adapted from [5, 8, 61-64].

1.2.3 Assembly and regulation of γ-secretase

Numerous studies have shown that γ-secretase is predominantly localized within ER-Golgi

compartments [41, 50, 65-67], although small amounts have been observed at the cell surface

where activity may occur [27, 68]. However, the precise steps by which the complex is

assembled within the secretory pathway have remained elusive [41, 42, 69]. Most models agree

that the formation of an early subcomplex between anterior pharynx defective 1 and nicastrin

within the ER is a first step in stabilizing presenilin holoprotein [28, 66, 67]. The remaining

steps, however, are more ambigous. Two models have been proposed for incorporation of

β

γ

z e

a

e

γ

A I C

D

A I C

D

Amyloidogenic

pathway

Non-amyloidogenic

pathway

Extracellular

hAPP mNotch1

S1 (Furin)

S2 (TACE)

S3

(γ)

S4

(γ)

N β

N I C D

A β

p 3

N E X

T

C99 C83

APPsβ APPsa

7

PSEN and PSENEN into an APH1-NCSTN subcomplex. The first model suggests that PSEN

holoprotein joins an APH1-NCSTN subcomplex to form a trimeric intermediate that later binds

PSENEN in the ER, and is then sorted out to the Golgi for complex activation [41, 65, 66],

similar to what has been observed in Drosophila [28, 50]. An alternative model proposes a

PSEN-PSENEN intermediate that joins an APH1-NCSTN subcomplex, generating intermediate

inactive subcomplexes comprising, for instance, PSENNTF-PSENEN and APH1-NCSTN-

PSENCTF [7].

Complex assembly involving multiple steps has also been shown to occur within post-ER

compartments, such as the intermediate compartment and cis- and trans-Golgi networks [42,

69]. This may also require numerous secondary binding factors that control transport, stability

and maturation. For instance, a molecule known as Retrieval to the Endoplasmic Reticulum 1

(Rer1) interacts with NCSTN [42] and PSENEN [70] in the intermediate compartment, or cis-

Golgi, to facilitate retrieval of unassembled components to the ER. Since presenilin enrichment

has been observed in COPI-coated vesicles, which traffic between ER and Golgi compartments,

there may be additional retrieval molecules involved in complex assembly [24, 54]. Additional

interactors have been identified, although their involvement in trafficking, maturation or activity

of the complex has not been explored in depth. Recently, the transmembrane glycoprotein

CD147 was identified as an integral regulatory subunit of γ-secretase that is required for down-

regulation of Aβ production [71]. Another transmembrane protein known as TMP21,

belonging to the p24 family of cargo retrieval receptors within ER-Golgi compartments,

modulates γ-secretase activity towards specific sites of APP cleavage [72]. Moreover, PSEN1

interacts with phospholipase D1, a phospholipid-modifying enzyme that recruits γ-secretase to

the Golgi/trans-Golgi network and negatively regulates Aβ generation [73]. A crystallized

structure of the complex may reveal the precise components or subdomains that contribute to its

activity, although with approximately 19 transmembrane domains, this may prove difficult. In

addition, the recent prevalence of additional γ-secretase cofactors suggests that assembly and

trafficking of the complex is highly regulated, and may involve other putative modulators.

1.2.4 The spatial paradox

While γ-secretase assembly occurs predominantly in the secretory pathway, it remains

unclear where γ-secretase activity is required to cleave its substrates. The relationship between

γ-secretase activity and the subcellular localization of its substrate proteins has been rigorously

8

examined. Localization of γ-secretase has been predominantly observed in ER-Golgi

compartments, and in minute amounts at the cell surface. However, substrate proteolysis is

believed to occur exclusively at the plasma membrane or in late endosomal compartments [24,

74-76]. It was therefore puzzling that only low levels of γ-secretase were detected at or near the

cell membrane [26, 27]. Similarly, APP processing is nearly absent in the ER, where the

majority of presenilin is detected [24, 77]. This discrepancy between presenilin localization and

γ-secretase activity has been referred to as the ‘spatial paradox’ [24, 41, 42]. Accumulating

evidence has now shown that internalization of APP at the cell surface is required for

amyloidogenic processing. That is, mutagenesis studies that disrupt a putative C-terminal

endocytic sequence abolish processing of APP [78]. Additionally, γ-secretase localization has

been observed in cholesterol-rich microdomains, known as lipid rafts, at post-Golgi and

endosomal compartments [79, 80], where the majority of APP proteolysis by γ-secretase is

believed to occur [81]. To complicate this, Aβ42 processing has been observed in pre-Golgi

compartments, possibly in the ER [76, 82], and Aβ40 generation has been detected in post-Golgi

and endocytic compartments [77, 82]. Thus, it has been proposed that two pools of presenilin

may exist in the cell, one distributed in pre-Golgi compartments in the ER and COPI-coated

membranes, and another in post-Golgi compartments at the cell surface and in endosomes [77].

Reconstitution of the active γ-secretase complex, by mixing extracts of purified ER and post-ER

compartments, may provide more definitive molecular insights into its activity [24].

1.3 Targets of the γ-secretase complex

There are over 30 known γ-secretase targets, including the well-known Notch and APP

substrates [8, 54, 83]. Other targets include Delta, Jagged or Serrate, the N- and E-cadherins

and ErbB4 [8]. Since null mutations in presenilin cause Notch-related defects in flies and mice

[84, 85], I will discuss the Notch signalling pathway below.

1.3.1 The role of Presenilin-dependent γ-secretase activity in Notch signalling

Several studies have shown that Presenilin is an integral member of the Notch signalling

pathway. For instance, Presenilin loss-of-function mutants have been shown to contribute to an

array of classic Notch-related phenotypes in mice [85, 86], Drosophila [84, 87, 88] and C.

elegans [89, 90]. Similarly, Presenilin as part of the γ-secretase complex is essential for the

9

final intramembrane cleavage of the Notch receptor, which results in release of an intracellular

domain required for nuclear signalling events (as described in Fig. 1.2). Depletion of individual

components of the γ-secretase complex destabilizes the active complex [28, 51, 54], and Aph-1,

Nct and Pen-2 loss-of- function mutants display defects in γ-secretase-dependent Notch

processing and signalling activities in Drosophila [48, 67, 91] and C. elegans [46, 92, 93].

Here, I describe the key events for activation of Notch that require Psn-dependent activity.

1.3.2 Summary of the Notch signalling pathway

The ability for multicellular organisms to establish complex biological patterns requires

temporal and spatial control of cell-cell communications [94]. In this way, cells respond to one

another to modulate intercellular and intracellular fates and behaviours. Notch signalling is

essential to Metazoa, primarily for cell-cell communication events that allow proper fate choices

for differentiation, cell death and proliferation [95, 96]. The core pathway comprises the Notch

receptor, DSL ligands (Delta/Serrate/Lag-2), CSL DNA-binding proteins

(CBF1/RBPjk/Suppressor of Hairless/Lag-1) and HES target genes (Hairy/Enhancer of split)

(Fig. 1.3; [97]). Vertebrates encode four Notch receptors, referred to as Notch 1 – 4, whereas

flies possess a single Notch gene. Synthesis of the receptor in the secretory pathway involves a

furin cleavage at the S1 site generating a stable heterodimer that is presented at the cell surface

in mammals, although an intact Notch receptor is predominantly detected at the plasma

membrane in flies [98]. Notch activation by DSL ligand binding may confer a conformational

change that allows access to the S2 cleavage site by metalloproteases of the ADAM family, such

as TACE in vertebrates and Kuzbanian in flies [99, 100]. The large ectodomain is shed during

this process, and the remaining fragment, known as NEXT, is retained at the plasma membrane

[61]. This fragment is recognized by an active Psn-dependent γ-secretase complex, and

cleavage occurs within the transmembrane domain at the S3 and S4 sites [61]. This is believed

to occur at the membrane or in endosomal compartments [99, 101]. The γ-secretase cleavage

releases the Notch intracellular domain (NICD) that translocates to the nucleus. In the nucleus,

NICD binds to CSL proteins and other transcription factors to regulate gene expression of target

genes required for Notch-mediated signalling.

Disrupted Notch signalling produces a neurogenic phenotype in flies, comprising

overgrowth of the nervous system at the expense of epidermis. In addition to Notch, mutations

10

Figure 1.3 Schematic of the Notch signalling pathway. In vertebrates, Notch (blue vertical bars) binding to ligand (Delta is shown here) at the cell surface elicits ectodomain shedding. This catalyzes γ-secretase processing of Notch within the transmembrane domain in the signal-receiving cell. The NICD is released, which translocates to the nucleus and associates with the transcriptional cofactor known as mastermind (mam) and CSL DNA-binding transcription factors. This complex activates target gene expression, such as Hairy/Enhancer of split (HES). Adapted from [97].

in the neurogenic genes, Notch, big brain, mastermind, neuralized, E(spl), Delta and almondex

give rise to similar phenotypes [102-108]. Notch function has been implicated in almost all

developmental processes throughout Metazoan development [94, 109]. It is also one of the rare

genes that demonstrates haploinsufficiency, since a single copy of the gene, when mutated,

induces mild Notch defects. As mentioned, processing of the Notch receptor is highly regulated,

requiring successive cleavage events for activation of downstream target genes. I will explore

developmental paradigms that require Notch signalling in the sections to follow.

Extracellular

S2 (TACE)

S3, S4 (γ-secretase)

Endoplasmic

reticulum

Nucleus

Golgi

S1 (Furin)

CSL

mam

Target gene expression (HES)

NICD

NICD

Delta

NOTCH

NOTCH

Signal-sending cell

Signal-receiving cell

11

1.3.3 Involvement of Notch signalling in lateral inhibition

Notch-mediated lateral inhibition has been shown to play an important role in the

development of the Drosophila adult peripheral nervous system. Briefly, sensory structures

arise from a group of equipotent cells during the development of multicellular organisms. A

classic example is the process of lateral inhibition, which involves the segregation of neural and

epidermal precursor cells of the peripheral nervous system [110]. In third instar larval wing

discs, equipotent groups of cells, referred to as proneural clusters, were shown to express

proteins encoded by the achaete-scute complex of proneural genes (Fig. 1.4 A). These basic

helix- loop-helix transcription factors accumulate over time in cells that adopt the neural fate,

such as sense organ precursors (SOP) of larval wing discs (Fig. 1.4 B; [99, 111, 112]) or

neuroblasts in the embryo [113], and in turn a similar fate is suppressed in surrounding cells.

Notch signalling normally functions to restrict the neural cell fate choice in cells that surround a

single neural precursor, by repressing the expression of proneural genes [110]. Reduced Notch

signalling at key developmental stages causes all cells of a proneural cluster to express high

levels of proneural proteins, remanding them to a default neural cell fate [94].

An adult sensory organ is formed from five distinct cell types, known as shaft, socket,

glial, neuron and sheath, which are derived from asymmetric cell divisions within individual

SOPs (Fig. 1.4 C). The surrounding cells are restricted from developing into neuronal cells, and

adopt an epidermal cell fate. Lateral inhibition therefore establishes the pattern of adult sensory

bristles in Drosophila, for instance, in the adult notum [110]. In the fly eye, specification of the

R8 founder cell of each ommatidium also requires lateral inhibition [95]. In mammals, lateral

inhibition has been shown to be involved in hair cell maintenance of the ear [114-116].

1.3.4 Notch activity in other signalling paradigms

Notch activity is also involved in boundary formation (Fig. 1.5 A). In the wing imaginal

disc, Notch signalling is concentrated at the dorsal/ventral boundary to establish patterning of

the dorsal and ventral compartments and to define the presumptive wing margin [117]. The

Notch ligands, Serrate and Delta, interact with Notch and restrict Notch activity to the

dorsal/ventral boundary during wing disc development [117]. Notch signalling then induces

expression of Wingless, through a process known as inductive signalling, which directs Serrate

and Delta expression outside of the boundary as part of a dynamic feedback loop [118]. Finally,

Notch activates expression of the transcription factor known as Cut at the presumptive wing

12

Figure 1.4 Notch-dependent lateral inhibition specifies bristle sense organ development. (A) Expression of proteins of the achaete-scute complex within proneural clusters (dark grey; left panel) confers their neural identity. Lateral inhibition ensures that proneural gene expression is maintained in single cells that will commit to a sense organ precursor (SOP) cell fate (dark grey; middle and right panels) and inhibit surrounding cells from adopting this fate (light grey; middle and right panels). (B) Fate map of an imaginal wing disc indicating sense organ precursor cells (SOP; dark grey circles) and the corresponding bristle sites of an adult heminotum (open circles). Adapted from [119]. Abbreviations are as follows: a, anterior; p, posterior; DC, dorsocentral; NP, notopleural; PA, postalar; SA, supraalar; SC, scutellar bristles, respectively. (C) Asymmetric cell division and maturation of individual SOPs (dark grey) into the five known specialized cell types of adult sense organ bristles, known as glia (brown), neuron (light grey), sheath (light blue), shaft (yellow) and socket (light green). Schematic of an adult sense organ showing the cell fates, and a representative WT adult notum bristle. The internal cells, glia, neuron and sheath are not visible whereas the external cells, shaft and socket, can be observed. Enhanced Notch signalling can result in excessive neuronal cell specification, at the expense of socket and shaft cells, and reduced Notch activity can give rise to additional sense organs (not shown; [94]). Schematics adapted from [94, 97].

margin, inhibiting ligand expression at the boundary and ensuring proper wing growth and

patterning [118, 120]. Loss of Notch signalling at the wing boundary results in classic wing

‘notching’ phenotypes, compared to WT (Fig. 1.5 B and C).

pI

pIIa

pIIb

pIIIb

Glial Cell

Neuron

Sheath

Shaft

Socket

A

B

Equivalent group of cells Restricted cell fates Lateral inhibitory specification

C

NP SA

DC SC

PS PA aSC

pPA

aDC

PS

pSA

aNP

pNP

pSC

pDC

aSA

aPA

13

Figure 1.5 Notch activity specifies cell fates through the process of inductive signalling. (A) Notch signals from a group of cells (red) to neighboring non-equivalent groups of cells (white) to induce a new cell fate, or adopt a new behaviour (dark grey). Adapted from [94, 97, 109]. (B) Inductive signalling in the larval wing disc promotes a smooth wing margin in WT adult flies. (C) An adult wing displays notching (arrowhead points to a representative notch) at the distal wing tip in heterozygotes of the Notch deficiency, Df(1)N-8/FM7. Overgrowth of the wing is observed in Notch gain-of-function alleles (not shown).

The Drosophila eye has also been shown to require Notch signalling at multiple steps of

photoreceptor cell specification, and for assembly of ommatidia [95, 96, 121, 122]. The normal

adult eye exhibits approximately 800 photoreceptor units, known as ommatidia, each with its

own accessory cells. In third instar larvae, a field of equipotent epithelial cells undergoes

differentiation, marked by a morphogenetic furrow that progresses across the field, and specifies

cells posterior to the furrow to become ommatidia precursors. Subsequent cell division and

apoptosis are required to specify proper ommatidial cell fates [95]. Individual ommatidia

comprise eight photoreceptor neurons, and associated cone cells, pigment cells and

interommatidial bristles [95]. Notch signalling coordinates with numerous other factors to

initiate the morphogenetic furrow and to control assembly of ommatidial preclusters by

specifying the R8 founder cell. Consistent with this model, Notch loss-of-function during

proneural enhancement inhibits R8 specification [123] resulting in neurogenic defects [124], and

fails to initiate furrow progression [125]. Loss of Notch signalling during lateral inhibition also

results in neurogenic defects, although increased numbers of R8 cells have been observed [123].

Therefore, Notch has been shown to confer multiple signals during photoreceptor development.

Notch-mediated signalling also controls programmed cell death in the eye imaginal tissue,

which is required to remove superfluous cells in developing ommatidia. Loss of Notch activity

during eye development prevents cell death in the interommatidial lattice [126].

A

Inductive Signalling Induced Cell Fate

C

B

14

In addition to its roles during imaginal disc development, which have already been

addressed, Notch signalling is also required during oogenesis. In the germarium of adult

ovaries, a group of 16 interconnected germline cells is surrounded by somatic follicle cells, that

form an epithelium around individual egg chambers [127]. Multiple egg chambers at various

stages of development are then separated by stalk attachments, comprising an intact ovariole

(Fig. 1.6). Cooperative signalling between the Notch and JAK/STAT pathways is required to

specify follicle cell types and to establish the anterior-posterior axis of the oocyte in early stages

of oogenesis [128, 129]. One of the 16 germline cells is selected to become the oocyte,

occupying a posterior position of the egg chamber, likely due to DE-cadherin-mediated adhesion

between the ooctye and posterior follicle cells, and to Notch/Delta-JAK/STAT interactions in

the germarium [127, 130].

Figure 1.6 Schematic representation of the anterior portion of a Drosophila ovariole. A WT ovariole comprising a germarium of developing egg chambers, and stage 1 – 7 egg chambers. Nurse cells adjoin individual oocytes (red). Somatic follicle cells encase the egg chamber from stage 1 onward. Notch signalling is believed to be required for anterior-posterior (A-P) patterning of the oocyte in germline cysts that bud from the germarium, and during the switch from mitosis to endoreplication (mitotic-to-endocycle transition). Adapted from [127, 129, 131-133].

In the developing egg chamber, Notch signalling has been shown to mediate a mitotic-to-

endocytic transition in follicle cells at stage 6, when the cells switch from a mitotic cell cycle to

an endoreplication cycle when the cells are no longer dividing [132]. In successive stages, the

Notch receptor is cleared from the apical membrane [128, 132, 133]. Notch also controls stem

cell formation and maintenance in the germline [134]. Defects in Notch expression have been

Germarium Nurse cell Oocyte

Follicle cells

Egg chamber

STAGE: 1 2 6 4 7

Stalk

Mitotic-to-endocycle transition A-P patterning of the oocyte

15

shown to result in a loss of germline stem cells, fusion of egg chambers, loss of anterior-

posterior patterning and prolonged mitosis at the expense of endocycles, leading to

overproliferation of follicle cells [128, 132-134].

1.3.5 Notch activity during hematopoiesis

There are many parallels between vertebrate blood cell development and Drosophila

hemocyte development, and therefore the fly has emerged as a valuable resource for insights

into how innate immunity may be regulated. Two successive waves of immune cell

differentiation, and functional conservation of signalling pathways and factors have been

observed in both systems [135, 136]. For instance, the Notch signalling pathway plays

important roles in crystal cell and lamellocyte specification in flies, since loss of Notch function

resulted in decreased levels of both cell types [137]. In vertebrates, Notch controls

hematopoietic stem cell generation and T-cell and B-cell lineage development [135, 137, 138].

Drosophila hematopoiesis occurs in two stages, one during embryogenesis and another in

larval development (Fig. 1.7; [136]). In embryos, hemocytes derived from the head mesoderm

migrate throughout the hemolymph [139], whereas in larvae, the lymph gland is the primary

source for hemocyte production [137]. Hemocytes give rise to three cell lineages that circulate

the hemolymph, macrophage- like plasmatocytes, crystal cells involved in melanization and

lamellocytes that differentiate upon parasitic infection [136, 140]. Plasmatocytes are the major

constituent of the hemolymph, comprising up to 94% of immune cells [136, 141, 142]. These

cells engulf apoptotic cells generated during metamorphisis of embryonic and pupal tissues, and

additionally remove pathogens in larvae and adults [135]. Plasmatocytes also express factors

that are involved in production and secretion of components of the extracellular matrix, such as

the collagen type IV protein, viking [135, 142, 143]. Crystal cells constitute approximately 6%

of the total population of hemocytes [136, 141]. These cells function in wound healing, and in

the melanization process, which facilitates innate immunity by secreting compounds that are

toxic to microorganisms [135, 136, 141]. A common parasite in Drosophila that is not easily

processed by plasmatocytes or crystal cells, are the eggs from female wasps that are deposited in

the hemolymph of young larvae [141]. The larvae respond to this invasion by rapid

proliferation and differentiation of lamellocytes, which are able to target and phagocytose large

invaders [136, 141, 142].

16

Figure 1.7 Drosophila hematopoietic cell lineage. A schematic representing the molecular regulation of hemocyte differentiation in embryos and larvae. Serpent (Srp) is expressed in all precursor cells, or prohemocytes. The majority (thick arrow) of Srp-expressing cells subsequently express Glial cells missing (Gcm), and differentiate as plasmatocytes. A small subset (thin arrow) of Srp-positive cells express Lozenge (Lz), and give rise to a fraction of Gcm-derived plasmatocytes, or differentiate as crystal cells in response to the Notch signalling pathway and U-shaped (Ush). In larvae, lamellocyte production requires the coordination between the Notch, Toll and JAK/STAT pathways. Adapted from [136, 137, 140, 144].

The accepted view so far is that most tissues, at many different levels and stages of

development, are affected by Notch signalling. The implication for this study is that multiple

Notch-related processes also depend on proper maintenance of Psn protein. These topics will be

addressed in the following Chapters.

1.4 Presenilin function independent of its γ-secretase activity

Recent studies have focused on the abundant intracellular functions of presenilin, linking

the protein to calcium regulation, cell adhesion, protein trafficking, signal transduction, tau

phosphorylation and transcription regulation activity [145]. Relevant to this study, is the role of

presenilin function in intracellular calcium homeostasis and apoptosis. Interactions between

presenilin and many known calcium regulators, including calsenilin and Sorcin and the calcium

release channel, Ryanodine receptor, are involved in calcium regulation in the cell [145-149].

Presenilin holoproteins also form passive ER calcium leak channels [150]. Alterations of

Prohemocyte

Crystal Cell (melanization)

Plamatocyte

(phagocytosis)

Srp

Lz

Ser/Notch

Gcm

Lamellocyte (encapsulation)

Lz

Ser/Notch

JAK/STAT

Notch

Toll

Ush

Em

bry

os

Larv

ae

Gcm

Plamatocyte

(phagocytosis)

17

intracellular calcium levels have been consistently linked to numerous clinical familial

Alzheimer’s disease (FAD) mutations in PSEN1 and PSEN2, in cell-based assays [151-153].

Moreover, presenilins have been shown to modulate aspects of intracellular apoptosis

regulation, since mutations in PSEN1 and PSEN2 associate with excessive pro-apoptotic

activities in AD [154]. I have already mentioned the prevalence of amyloid plaques,

neurofibrillary tangles and loss of neuronal tissues in AD patients, but it is still unclear if these

are causative for the disease. Accumulating evidence suggests that defects in calcium signalling

facilitate AD pathology before development of detectable markers or cognitive impairment

[151, 155]. Since calcium function has also been implicated in neuronal viability and apoptosis,

calcium has become a significant player in current AD research. I will summarize the roles of

presenilin function in calcium homeostasis and apoptosis, and briefly highlight the role of

calcium dysfunction in AD in the following sections.

1.4.1 Intracellular calcium homeostasis, regulated by presenilin, is defective in AD

Regulation of calcium stores within the cell is crucial to many processes that range from

transcription of genes to synaptic plasticity [155]. Extracellular calcium enters the cytosol

through gated channels at the plasma membrane, and from internal ER stores via two types of

calcium release channels, known as the inositol-1,4,5-trisphosphate receptor (IP3R) and

Ryanodine receptor (RyR) [151, 156]. Endogenous presenilin function has been shown to

contribute to cytosolic calcium levels, since it forms an ER calcium leak channel independent of

critical aspartate residues required for its γ-secretase activity. Its role in calcium regulation does

not require γ-secretase activity, since APH1 null mice show normal ER calcium release [150].

Thus, presenilins appear to contribute to AD neurodegeneration through at least two separate

mechanisms, involving their role as a critical component of the γ-secretase complex, as already

discussed, and another function involving calcium signalling in the ER, which will be further

explored below.

Alterations in calcium levels observed in both early and late cases of AD led to the

calcium hypothesis, which states that sustained calcium-induced injuries contribute to

neurodegeneration [157]. Calcium has since been shown to associate with many known AD risk

factors. For instance, exogenous soluble Aβ oligomers caused a rapid influx of calcium from

extracellular and intracellular stores in the cytosol of mammalian cells, resulting in membrane

disruption and cell death [21, 158]. Secreted APP molecules (APPs) however, are believed to

18

be neuroprotective, stabilizing cytosolic calcium levels and attenuating the pro-apoptotic effects

of mutant PSEN1 [159]. Whether calcium disruption affects APP processing, or defects in APP

proteolysis lead to aberrant calcium signalling remains unclear. FAD-associated mutations in

PSEN1 and PSEN2 and loss-of- function mutations show elevated calcium release from ER

stores long before extracellular Aβ pathologies become apparent [21, 151]. This may be due, in

part, to an increase in cytosolic calcium levels that promotes β-secretase cleavage of APP, and

subsequent γ-secretase activity may induce intracellular Aβ42 production and plaque deposition

[160]. In contrast, γ-secretase- inhibition and loss-of- function studies in murine fibroblasts

showed reduced Aβ formation prior to reduced ER calcium content, and surprisingly this defect

was rescued by addition of AICD suggesting a physiological role of AICD in calcium

maintenance [161]. Thus, although much research has provided insights into the possible roles

of calcium in neurotoxicity, the precise mechanisms of calcium-induced injury to the cell remain

undetermined.

Presenilins have thus emerged as important players in the calcium signalling pathway,

given their functions in maintaining ER calcium stores [21]. Numerous studies have shown

disrupted calcium signalling in fibroblasts from FAD patients [162], in PSEN1 and PSEN2

mutant Xenopus oocytes [163, 164], in PSEN1 mutant mammalian cells [165] and in neurons

from mutant PSEN1-expressing mice [152, 166], involving both IP3R and RyR ER calcium

channels, and distinct from calcium entry at the plasma membrane. Another study has shown

that PSEN1 mutations resulted in increased levels of the ER-specific calcium release channel,

RyR, and calcium release in primary neuronal culture [167]. PSEN1-deficient mice also

exhibited disrupted calcium homeostasis that increased neuronal cell vulnerability towards

oxidative stress, apoptosis and metabolic insults, and led to cell death [146, 168]. The

involvement of Aβ and presenilin function within the calcium signalling pathway highlights a

tight link between calcium homeostasis and Alzheimer’s disease. This may lead to therapeutic

strategies to help maintain neuronal calcium homeostasis [146]. Additional analysis using

conditional presenilin knock-out experiments may help to determine whether the multiple roles

of presenilin are mutually exclusive, but will not be addressed further here.

1.4.2 The role of presenilin in the apoptotic signalling pathway

Apoptosis is a mechanism of programmed cell death that is normally required during

development to ensure proper cell numbers, cell types and morphology. For instance, apoptosis

19

helps to regulate ommatidial cell- fate specification in the developing eye disc of Drosophila

[95]. Apoptosis also plays an underlying role in human disease, such as Alzheimer’s disease.

For example, the effects from neurotoxicity, oxidative stress, disrupted mitochondrial function,

Aβ peptides and increased intracellular calcium levels can lead to cell death [155, 169-171].

Induction of apoptosis alters mitochondrial permeability, activating caspase-cleavage events in

the apoptotic pathway that lead to cell death [155, 170, 172].

Several studies in cultured cells have indicated that the C-terminal domains of PSEN1 and

PSEN2 are substrates for caspases, such as caspase-3, suggesting that presenilins may be

involved in apoptosis regulation [173, 174]. For instance, FAD-linked PSEN1 mutations

enhanced the apoptotic effects resulting from oxidative stress and caspase activation in primary

cultured neurons from the cortical brain regions of transgenic mice [175]. PSEN1-deficient

neurons also exhibited increased levels of apoptosis and caspase-3 activation leading to neuronal

degeneration [176]. Similarly, phosphorylation of PSEN1 at a site that is recognized by

caspases reduced the progression of apoptosis [177], which may involve interactions between

presenilin, the lipid kinase, PI3K, and its downstream factor, the serine-threonine kinase Akt, as

part of a cell signalling pathway that mediates survival of cerebellar neurons [178]. Akt

function reduces the activities of known apoptotic factors, including members of the Bcl-2

family of cell death regulators and caspases [176, 178]. Presenilin FAD mutations have also

been shown to activate the apoptotic factor, caspase-3, and impair the neuroprotective role of

PSEN1 in the PI3K/Akt signalling pathway, leading to increased apoptosis in murine primary

neuronal cells [176]. It would be interesting to compare the levels of apoptosis in mature, post-

mitotic neuronal cells with apoptotic levels in developing neurons.

Importantly, presenilins have been shown to form multimeric complexes with members of

the Bcl-2 family of proteins, including Bcl-2 and FKBP38. Bcl-2 is an anti-apoptotic factor that

binds to FKBP38, a mitochondrial- localized immunophilin, thereby inhibiting intracellular

apoptosis [179, 180]. FKBP38 is unique amongst the FKBP-type immunophilins, since it shows

neither an affinity for the FKBP-inhibitor known as FK506 nor peptidyl-prolyl cis-trans

isomerase activity, which defines members of this family [179]. PSEN1 and PSEN2 form

protein complexes with FKBP38 and Bcl-2 in vivo, independent of γ-secretase activity, targeting

them to ER/Golgi compartments and promoting their degradation, thereby rendering the cell

susceptible to apoptosis [154]. Presenilin FAD mutations enhanced the apoptotic susceptibility

of primary cultured neuronal cells, by targeting more Bcl-2 into ER/Golgi fractions compared to

WT presenilins, and caspase- induced presenilin C-terminal fragments abrogate the interaction

20

between PSEN1 and FKBP38, resulting in decreased intracellular sensitivity to apoptosis [154].

Apoptotic stimuli may therefore accelerate AD-related pathology, mediated in part by the effects

of FAD mutations on apoptotic signalling from mitochondria. Taken together with a

requirement for presenilin in γ-secretase activity, these data provide evidence of presenilin

pleiotropic functions in the cell. The interaction between Psn and another member of the FKBP

family of proteins, known as FKBP14, will be discussed in depth in Chapter 3.

1.5 Overview of FKBPs

Immunophilins comprise a family of peptidyl-prolyl cis-trans isomerases (PPIases),

characterized by their ability to catalyze the cis-to-trans isomerization of peptide bonds [181].

Subsequently two types of PPIases were identified as binding targets for immunosuppressant

drugs, namely cyclophilins which bind cyclosporin A, and the FK506-binding proteins (FKBPs)

that bind FK506 and rapamycin [182, 183]. Humans encode at least 15 FKBPs, whereas 8

FKBPs have been identified in flies [57, 182, 184]. Prototypical human FKBPs, such as

FKBP12, FKBP13 and FKBP14 (FKBP22) contain just one PPIase domain whereas other

FKBPs possess multiple PPIase domains, in addition to tetratricopeptide (TPR) motifs which

may function as protein interaction domains (Fig. 1.8 shows the structure of 6 FKBPs that are

relevant to this study; [57, 184, 185]). FKBPs have been identified in a wide range of organisms

and tissue types, and are expressed throughout development, suggesting that they may play

general roles in the cell [181, 183, 186]. However, more recent data implicate specific functions

for a wide variety of FKBPs, which will be discussed below.

1.5.1 FKBP function regulates intracellular calcium signalling

The first human FKBP to be identified was a 12 kDa FK506 target protein known as

FKBP12 [187, 188]. Subsequently, it was identified as a constitutive binding partner of the

calcium release channel, Ryanodine receptor, in skeletal and cardiac muscle tissue [189-191]. A

single channel comprises four Ryanodine receptors, each bound to one FKBP12 molecule that is

believed to stabilize the interaction between the four monomers [190, 192]. Removal of

FKBP12 from this complex by drug competition or deletion, caused an increase of Ryanodine

receptor gating capacity and subconductance states in planar lipid bilayers [191], Spodoptera

21

Figure 1.8 Domain structure of 6 characterized human FKBPs. FKBP12 (also known as FKBP1A) defines the minimal element required for PPIase activity, known as the FKBP_C domain (black box), or PPIase domain. FKBP13 (FKBP2) encodes an additional N-terminal signal peptide (S; grey box) and a C-terminal ER retention motif, RTEL. FKBP22 (FKBP14) and FKBP23 (FKBP7) exhibit similar structures, displaying N-terminal signal peptides (S; grey boxes), an individual FKBP_C domain (black boxes), C-terminal EF-hand calcium binding motifs (EFh; white boxes) and C-terminal ER retention sequences, HDEL. This ER retention sequence is a variation of the well-characterized ER retention sequence, known as KDEL, in the ER-localized chaperones PDI and BiP [193, 194]. In addition to the FKBP_C domain (black box) in FKBP38 (FKBP8), TPR domains (black diamonds) are exhibited at the C-terminus. FKBP51 (FKBP5) exhibits multiple FKBP_C domains (black boxes), and C-terminal TPR motifs (black diamonds). Adapted from [184]. FKBPs were originally named according to the molecular weights of their encoded proteins, as shown. Recent gene names are shown in brackets.

frugiperda cells [192] and FKBP12-deficient mice [189], resulting in increased intracellular

calcum levels [192]. FKBP12 also binds with the IP3R calcium release channel, and presumably

modulates the phosphorylation status of the channel by recruiting the phosphatase calcineurin to

the complex [195]. Dissociation of FKBP12 from IP3R by FK506 treatment resulted in

increased calcium flux in rat cerebellar microsomes [196].

Another FKBP molecule involved in intracellular calcium signalling, known as FKBP51,

localized to murine T-cells and is widely distributed in humans [197]. Extensive studies in cell-

based systems have shown that FKBP51, its paralog known as FKBP52, heat shock protein 90

(Hsp90) and other factors are involved in steroid receptor transcriptional activity [198-200].

FKBP51 and FKBP52 possess two FKBP-like domains, one that exhibits PPIase activity, and

another which may exhibit chaperone function [182]. Similarly, ER-localized FKBP14

orthologs in yeast, known as FKBP22, display functionally independent PPIase and chaperone

activities [201], and ER-localized FKBP23 exhibits calcium binding mediated by C-terminal

FKBP23

FKBP22

(FKBP14)

FKBP12

FKBP13

FKBP38

FKBP51

HDEL S FKBP_C EFh EFh

S FKBP_C EFh

RTEL S

FKBP_C

FKBP_C

FKBP_C

FKBP_C

FKBP_C

TPR TPR TPR

TPR TPR TPR

HDEL EFh

22

EF-hand sequences [202]. Calcium ions are important to proper ER function, and the presence

of calcium-binding proteins, such as FKBPs, in the ER indicates possible functions in protein

sorting, folding and degradation [202].

1.5.2 The role of FKBPs during the unfolded protein response

PPIase protein folding activity was originally shown in an assay developed by Fisher et al

[203]. Briefly, a synthetic peptide known as N-Suc-Ala-Ala-Pro-Phe-p-nitoanilide was cleaved

by chymotrypsin only when Ala-Pro was in a trans configuration [204]. Approximately 10% of

these peptide bonds exhibited cis conformations, and the rate- limiting step for proteolysis was

observed to be a cis-to-trans isomerization of the bond performed by PPIases [186, 204]. The

mechanism whereby FKBPs catalyze cis-trans isomerization of prolyl bonds, is to bind and

lower the energy of an unstable twisted-amide intermediate conformation [182]. Thus, it was

shown that PPIases, and in particular FKBPs, can accelerate the prolyl isomeration of protein

substrates essential for protein folding.

Several other studies have implicated ER-localized FKBPs as integral to protein folding,

trafficking and assembly. For instance, FKBP13, which shares 43% homology to FKBP12,

localizes to the ER and is upregulated during the unfolded protein response in mammalian cell-

based assays [205-207]. It was also shown that the yeast ortholog of FKBP13, known as Fkb2,

mediates upregulation of the protein in response to unfolded proteins in the ER through a 21 bp

unfolded-protein-response element located 5’ to the coding region, suggesting a role in protein

trafficking in the ER [208]. Since expression of chaperones in the ER is induced in response to

elevated levels of unfolded proteins, this also suggests that FKBP13 may exhibit chaperone-like

activities.

1.5.3 FKBP14 localizes to the endoplasmic reticulum to facilitate protein folding

The ER is a specialized compartment for the proper synthesis and folding of proteins prior

to secretion and transport of proteins to downstream organelles [209]. Protein folding is highly

regulated, involving the chaperone systems known as the calnexin/calreticulin system and the

BiP system that independently recognize unfolded proteins in the ER and promote protein

folding through a series of substrate binding and release steps [209, 210]. The FKBPs have

23

recently emerged as putative members of these systems, functioning as folding catalysts that are

involved in protein folding in the ER [201].

An important FKBP to this study is a highly conserved protein known as FKBP14, or

FKBP22. The discrepancy in nomenclature reflects the wide range of terminology shared

amongst taxonomic groups. Orthologs in yeast and bacteria are referred to as FKBP22 [211-

213], although orthologs in mammals, known as FKBP14, have been less extensively studied

[214]. In yeast, FKBP22 proteins dimerize in the lumen of the ER, mediated by the C-terminal

domain [201]. A recombinant FKBP22 N-terminal domain exhibited PPIase activity, and

interacted with a well-characterized Hsp70 chaperone known as BiP, in the ER [201].

Interestingly, an FKBP22-BiP complex enhanced the PPIase- independent chaperone activity of

full- length FKBP22, since the complex prevented aggregation of unfolded proteins [201].

FKBP22 has subsequently been shown to associate with other chaperones and folding catalysts,

including another Hsp70 chaperone known as Grp170, and the most abundant ER-localized

disulfide isomerase, known as PDI [209]. This demonstrates a highly regulated chaperone

network in the ER, possibly to maintain protein quality control, and is consistent with reports

from mammalian cell-based assays [209, 210, 215]. A bacterial FKBP22 ortholog has also been

shown to dimerize, although this was mediated through the N-terminal domain, and its C-

terminal domain exhibited PPIase activity [216]. I will show in the following chapters that an

FKBP14 ortholog in flies is essential for development, and may be involved in protein folding

activity.

Identification of FKBP14 binding targets in Drosophila may provide further insights into

its physiological roles, and potential functions in protein folding, trafficking and signal

transduction. To further examine its role in development, we can perform mutational analysis of

FKBP14 (described in Chapter 3), and examine the effects of FKBP-specific inhibitors such as

FK506, on FKBP14 function (Chapter 5). I will explore the effects of FK506 on FKBP function

in detail below.

1.6 Effects of the immunosuppressant, FK506, on FKBP function

An immunosuppressant drug widely used by organ transplant patients, known as FK506

(Tacrolimus, Prograf), reduces the immune response thereby lowering the risk of organ

transplant rejection. This fungal macrolide was initially discovered in the fermentation broth of

24

Streptomyces tsukubaensis bacterial culture [217]. It exhibits strong immunosuppressant

properties through disruption of calcineurin-specific processes in T lymphocytes, with greater

potency compared to cyclosporin A, another immunosuppressant drug that targets cyclophilin-

type immunophilins (discussed above), and therefore, the use of cyclosporin has rapidly given

way to FK506 treatment [218, 219]. FK506 can, however, display significant neurotoxic and

nephrotoxic side effects in animal studies [219, 220]. However, trials using FK506 as a rescue

therapy in human liver transplant patients showed that FK506 had only minor side effects [219].

Currently, FK506 has been approved by the FDA for extensive use as an immunosuppressant

agent following many transplantation treatments, including liver, bone marrow, lung and kidney

transplantations in adults and children [218, 219, 221]. An understanding of the

immunosuppressive properties of FK506 began with the discovery of its cytosolic binding

target, known as FKBP12 [188, 222]. Since this discovery, FK506 has been shown to bind to

other FK506-binding proteins, or FKBPs, although specific affinities have yet to be determined.

I discuss the effects of FK506 on FKBP14 function in Chapter 5.

1.6.1 FK506 immunosuppression elicits FKBP12 gain-of- function during T-cell inhibition

FK506 has been shown to bind to a catalytic pocket in the PPIase domain of FKBP12,

inhibiting its activity, and subsequently other FKBPs have displayed varying affinities for the

drug [182, 186, 223, 224]. The biochemical mode of action involves an FKBP12-FK506

complex that interacts with calcineurin, a calcium-activated phophatase, inhibiting its

phosphatase activity, and resulting in an increase in phosphorylated substrates [195]. One of

these substrates is a transcription factor known as nuclear factor of activated T cells (NFAT),

that in its phosphorylated form, accumulates in the cytoplasm and is unable to stimulate

interleukin-2 production in the nucleus [182, 195]. Interleukin-2 plays a prominent role during

T-cell differentiation and proliferation, thus its inhibition by FK506 causes an

immunosuppressive action in T-cells [182, 195, 219]. An FKBP12-FK506 gain-of- function

model was postulated since FK506 treatment was shown to impart a new immunosuppressive

function to FKBP12 even at minimal intracellular concentrations [219, 225].

It is believed that FKBP12, its paralog FKBP12.6 and FKBP51 are the only intracellular

FKBP molecules responsible for the immunosuppressive activities of FK506 [202, 226].

FKBP51-FK506 complexes, however, showed significantly reduced potency compared to what

has been observed with FKBP12 or FKBP12.6 in cell-based assays [197, 222]. Since FKBP12

25

also exhibits calcium signalling activity, as already discussed, FK506 treatment may impart

multiple effects in the cell, for instance, by dissociating FKBP12 from intracellular calcium

channels, inducing intracellular calcium release and possibly cell death [192, 195]. In turn,

determining the effects of FK506 treatment on other FKBPs will require further analysis.

1.6.2 FK506 treatment epidemiology

As mentioned, FK506 is a powerful immunosuppressant, and although toxic side effects

have been observed in animal studies, these are less common in human transplant patients [218,

219]. Since some patients exhibited side effects in response to high doses of FK506, such as

toxicity of the kidneys [220], research to derive less toxic FK506 analogs and combination

treatments with an antimetabolite drug known as mofetil has continued [218, 219, 227]. A

comprehensive study of transplant patients in the United States of America from 1994-2004

showed that 60% of kidney transplant patients were using a combination FK506/motefil

treatment for inhibition of the T-cell response immediately following transplantation, and 50%

continued up to 2 years following transplantation [218]. Similar statistics were observed in

pancreatic, liver and lung transplant cases, whereas 98% of intestine transplantation patients

were using FK506 alone [218]. In contrast, only 31% of heart transplantation patients were

using FK506 in combination with motefil treatment up to 2 years after transplantation [218],

although the physiological basis for this is unclear. Further research into the intracellular roles

of combination therapy will provide insights into the mode of action, and which signalling

pathways are affected.

To conclude, FKBPs play important roles in protein folding and in promoting protein-

protein interactions. Specific roles have yet to be determined for many of the currently known

FKBPs. Identifying endogenous interacting partners will provide critical insights into their

functions in vivo. Pharmocological inhibition may also provide additional insights into FKBP

functions, although as mentioned, FKBPs display varying affinities for the drugs. Therefore,

this may be useful in addition to in vivo studies. Finally, crystallized structures may also

provide insights into protein-protein interaction domains or catalytic site motifs.

26

1.7 Rationale

The main goal of my study was initially to define the role of Presenilin in development.

Psn exhibits multiple functions in vivo, as an aspartyl protease of the well-characterized γ-

secretase complex and as a calcium signalling factor in the ER. Extensive research in cell-based

assays, model organisms and mammalian systems, has demonstrated multiple presenilin-binding

partners. Thus, I was interested in isolating other putative binding partners using Drosophila as

a model organism, since it encodes a single Psn protein.

In Chapter 2, I describe a classic genetic modifier screen aimed at identifying Psn-

interacting genes in Drosophila. One of these genes, known as FKBP14, emerged as a strong

candidate that warranted further analysis. Characterization of FKBP14, with combined

mutational analysis and examination of its role in development, is discussed in Chapter 3. I

demonstrated that FKBP14 function is required in many tissue types for multiple developmental

processes, particularly during larval disc development. My analysis also determined that an

interaction between FKBP14 and Psn is required to maintain Psn protein levels, and to mediate

downstream Notch signalling activity. In Chapter 4, I discuss the endogenous roles of FKBP14

during embryonic hematopoiesis and in bristle sense organ development. In Chapter 5, I

describe the effects of the FKBP-inhibitor drug, FK506, on levels of the γ-secretase

components.

Finally, in Chapter 6, I provide analysis and conclusions of my data, and discuss future

Presenilin and FKBP14 analyses in Drosophila.

27

CHAPTER 2

A GENETIC SCREEN TO IDENTIFY GENES THAT INTERACT WITH PRESENILIN

IN DROSOPHILA MELANOGASTER

Data Attribution: All experiments were performed by D. van de Hoef with the exception of the

genetic screen, which was performed by D. van de Hoef, J. Hughes and I. Livne-Bar, and the

genetic interaction data (Figure 2 A – C) that was performed by K. Michno.

28

2.1 Summary

The multimolecular g-secretase complex is involved in cleavage of transmembrane

proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as Amyloid

Precursor Protein. Presenilins function within the catalytic core of g-secretase. Recent studies

show that in addition to Notch, numerous signal transduction pathways are modulated by

presenilins, including those involving intracellular calcium signalling. Thus, presenilins appear

to have diverse functional roles. To further understand presenilin function, we searched for Psn-

interacting genes in Drosophila, by performing a genetic modifier screen for enhancers and

suppressors of Psn-dependent Notch-related phenotypes. Here I describe 178 modifiers that

enhanced or suppressed dominant Psn phenotypes. These genes include members of the Notch

signalling pathway, signal transduction and protein translation genes, transcription factors and

genes involved in intracellular calcium homeostasis. Characterization of these genes will provide

valuable insights into Presenilin function and may also provide potential targets for Alzheimer’s

disease drug therapies.

2.2 Introduction

Insights into the mechanisms underlying Alzheimer’s disease have come from the analysis

of mutations in three genes that have been linked with early-onset autosomal-dominant familial

Alzheimer’s disease (FAD), known as presenilin-1, presenilin-2 and the gene encoding the

Amyloid Precursor Protein (APP). Mutations in these genes associate with only 50% of FAD

cases however, which in turn accounts for less than 5% of all Alzheimer’s disease (AD) cases

[4]. This suggests that other genes may act as causative or susceptibility factors in AD. Since

missense mutations in presenilin account for the majority of FAD cases, we focused on

identifying genetic modifiers of presenilin.

Presenilins belong to an evolutionarily conserved family of integral membrane proteins

that are present in Metazoa. They play a critical role in development by regulating the Notch

signalling pathway, which mediates numerous cell fate decisions in multicellular organisms

[99]. Presenilins function as part of the g-secretase complex that cleaves single-pass

transmembrane proteins including Notch, APP, Delta, Jagged, ErbB4, ephrin-B1 and the N- and

E-cadherins [8, 145, 228, 229]. In vertebrates, APP is sequentially cleaved by β- and γ-

secretases within its transmembrane domain, releasing Ab peptides that under normal

29

conditions, are 1 – 40 residues in length (Aβ40), and an APP intracellular domain (AICD) that

translocates to the nucleus [63, 230, 231]. Recent studies show that the AICD migrates to the

nucleus and interacts with the adaptor protein, Fe65, and the histone acetylase, Tip60 [230, 232,

233]. AICD also regulates transcription of the EGF receptor [234] and even its own precursor

[232]. Missense mutations in human presenilins alter APP cleavage, increasing levels of a

longer Ab isoform, known as Ab42, that aggregates extracellularly and forms senile plaques in

brains of Alzheimer’s disease patients [63, 235]. These plaques have been suggested to be a

primary cause of AD-related neurodegeneration.

In addition to their proteolytic roles, presenilins have been implicated in numerous cell

signalling pathways, including intracellular calcium signalling and EGFR regulation [234, 236-

238]. In Drosophila, a single Presenilin gene has been identified that shares structural

similarities with sel-12 in C. elegans and the human PSEN genes. Importantly, Drosophila do

not encode β-secretase, although they do express an ortholog of mammalian APP known as

Drosophila APPL that does not contain an Aβ domain [239-241]. Therefore, under endogenous

conditions, flies do not produce Aβ peptides and are unable to recapitulate some aspects of AD-

related pathology in humans. However, there are fly transgenic models that have been

developed for these purposes [239, 242-245]. For instance, expression of human Aβ42 in

transgenic flies caused neurodegenerative defects associated with progressive accumulation of

Aβ peptides, age-dependent learning defects and shortened lifespan [242-244], whereas Aβ40

expression only caused age-dependent learning defects [244]. Additionally, targeted expression

of human APP, β-secretase and Drosophila Psn FAD-associated transgenes in the compound

eye of Drosophila resulted in production of Aβ42 plaques, age-dependent retinal

neurodegeneration and shortened lifespan [245]. Interestingly, human APP derivatives were

shown to be processed by endogenous α- and g-secretase activity in Drosophila [239]. Related

to this, is analysis of a Drosophila model of human tauopathy, which recapitulates certain

aspects of the human condition including neurodegeneration, early death and

hyperphosphorylated Tau, thereby contributing to our understanding of neurodegenerative

disease [15]. Drosophila has thus emerged as a model to analyze mechanisms underlying

neurodegenerative diseases.

In flies, the active g-secretase complex is believed to comprise four proteins known as

anterior pharynx defective 1 (Aph-1), presenilin enhancer 2 (Pen-2), nicastrin (Nct) and

Presenilin (Psn) [31]. Presenilin is thought to function as the catalytic core of this complex.

Consistent with this model, vertebrate presenilin has been shown to belong to a family of

30

transmembrane aspartyl proteases and mutations within conserved aspartyl residues in presenilin

abolish g-secretase activity [35-37]. Moreover, photoactivated transition-state analog inhibitors

of aspartyl proteases covalently bind to presenilin and inhibit g-secretase cleavage of substrates

[38-40]. Presenilin is synthesized as a 50 kDa holoprotein that is rapidly processed by an

unknown protease to generate amino and carboxy terminal fragments required for function [32,

33]. Aph-1 has been shown to stabilize Presenilin holoprotein, whereas Nct is believed to create

a substrate docking site or to promote trafficking of the complex throughout the secretory

pathway [28, 31, 48, 50, 246]. Finally, Pen-2 is required for endoproteolysis of Presenilin

holoprotein and for stabilization of mature Presenilin fragments [28, 46, 51, 53, 247-249].

Whether a multimolecular complex composed of Presenilin, Nct, Aph-1 and Pen-2 is

sufficient to reconsititute g-secretase activity in vivo remains unclear, however, studies in yeast

[31], Drosophila [28, 67] and mammalian cell culture [249, 250] have demonstrated that co-

expression of all four proteins can reconstitute g-secretase activity. Other studies show alternate

regulation of Notch and APP proteolysis by g-secretase, and suggest that other co-factors may

be required to mediate the interaction of the complex with its targets in vivo [251]. Consistent

with these results, a 440 kDa complex in vertebrates composed of PSEN, NCSTN, APH1 and

PSENEN has been identified that is unable to dock APP efficiently, whereas a complex of

approximately 670 kDa with unknown additional components exhibited full g-secretase activity

[252]. These studies suggest that the active complex may include multimers of the core

components, or that other factors may interact with the g-secretase complex to regulate its

assembly, stability, or enzymatic activity. For example, a PSEN dimer has been identified that

forms a functional diaspartyl group at the interface of the dimer within the g-secretase complex

[253, 254]. Another study demonstrated that a g-secretase complex containing only one of each

component was sufficient for substrate cleavage [255]. Assembly of the g-secretase complex

may also involve retrieval or retention signals to ensure its proper stoichiometry as it is

transported throughout the secretory pathway. Recently, a retrieval receptor known as Retention

of ER proteins 1 (Rer1) has been shown to interact with vertebrate NCSTN and PSENEN to

negatively regulate g-secretase complex assembly in ER-Golgi compartments [42, 70].

Additional factors involved in g-secretase regulation include CD147 and TMP21 [71, 72].

Further analysis is required to determine if other factors are required for g-secretase assembly,

stabilization and activity.

31

Drosophila provides an excellent model system to search for candidates that interact with

specific components of the g-secretase complex. All of the known g-secretase members are

conserved in Drosophila [245]. Reduction in the activity of individual components using either

RNA-mediated interference or null mutations demonstrates a critical role for each component in

Notch and APP processing [28, 46]. Since the function of g-secretase is conserved in flies, it

was possible to carry out a genetic modifier screen to identify regulators of g-secretase, and

specifically, to isolate genes that are involved in Drosophila Psn function. We recovered 178

modifiers that enhanced or suppressed Psn-dependent phenotypes in the wing and notum,

including candidate genes that function within pathways that involve Psn activity, notably calcium

signalling and Notch signalling. Genetic analysis of model organsims can therefore aid in our

understanding of the mechansim of PSEN activity in Alzheimer’s disease and development.

Here, I describe numerous classes of genes that may be involved in Presenilin function in

Drosophila.

2.3 Materials and Methods

2.3.1 Drosophila genetics

Flies were maintained on standard media. Transgenic lines carrying UAS-psn transgenes

were previously described [56]. Recombinant lines were generated that expressed UAS-psn

transgenes at 29°C with either cut-GAL4 (wing) or pannier-GAL4 (pnr-GAL4; notum) drivers,

and were approximately 80% penetrant. These lines were used to screen a collection of

approximately 1600 P- or EP-element transposon lines mainly on the 2nd and 3rd chromosomes,

which encode for the majority of proteins in Drosophila [256]. In the presence of GAL4, UAS

sequences contained in the EP insertions may drive expression of downstream genes, or

alternatively, generate antisense RNA products thereby inactivating gene expression [257-259].

Conversely, P-element insertions lack UAS sequences and likely inactivate gene expression

[259]. The original Psn recombinant lines were lost after screening 1600 lines. Molecular

characterization and chromosome location data for all modifiers were obtained from the

FB2008_07 version of the Drosophila melanogaster database [260].

FKBP14D58 represents an imprecise excision of the EP-element, EP(2)2019, and

FKBP14D34 represents a precise excision of EP(2)2019, as described in Chapter 3. Psn cDNA

32

was amplified from the EST clone LD23505 (Berkeley Drosophila Genome Project) and

engineered to express a C-terminal Myc epitope. The resulting fragment was cloned into a

pUAST vector to generate UAS-Psn-Myc. Transgenic lines carrying UAS-Psn-Myc transgenes

were generated by J. Hughes, using standard P-element transformation procedures [261].

Independent insertions were balanced over SM5 or TM3 chromosomes. Recombinant lines were

generated that expressed pnr-GAL4/UAS-Psn-Myc in FKBP14D34 or FKBP14D58 heterozygous

genetic backgrounds at 29°C. The Calmodulin alleles, Cam6806 and Cam11356, were analyzed by

Kinga Michno (K. Michno) for their effects on cut-GAL4/UAS-Psn wing phenotypes at 29°C.

2.3.2 Drosophila stocks

The cut-GAL4 line has been previously described [56]. The pannier-GAL4

(P[GawB]pnrMD237/TM3, Ser; 3039) and w1118 (3605) lines were obtained from the Bloomington

Stock Center. Stock numbers are shown in brackets. The collection of P- and EP-element

transposons that were used in the genetic screen were obtained from the Bloomington Stock

Center or Szeged collection.

2.3.3 Immunoblot Analysis

Flies were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1%

SDS, 0.02 molar Tris-Cl pH 8.0, 0.137 molar NaCl, 10% glycerol, 2 uM EDTA) supplemented

with Complete EDTA-free protease inhibitors (Roche). Lysates were centrifuged at 10,000 g

for 15 minutes. SDS-polyacrylamide gel electrophoresis (10% SDS-PAGE) and immunoblot

analyses were carried out as described previously [262]. Supernatants were analyzed using

rabbit a-PsnNTF (1:1000; [56]) to detect endogenous Psn N-terminal fragments and ectopic Psn

full- length proteins, rat a-FKBP14 (1:2000; described in Chapter 3) to detect endogenous

FKBP14 proteins and mouse a-JLA20 (Actin; 1:1000; DSHB) to detect Actin as a loading

control. HRP secondary antibodies were used (Jackson ImmunoResearch; 1:10 000). All blots

were performed in triplicate. Quantitation was performed using a Fluorchem 8000 Gel

Documentation System and Alpha Innotech software (San Leandro, CA).

33

2.4 Results

2.4.1 Presenilin overexpression causes Notch-related phenotypes in the notum and wing

Presenilin function has been implicated in numerous intercellular and intracellular

signalling processes. To further understand the function of Drosophila Psn, we performed a

classic genetic modifier screen to identify genes that interact with Psn. Specifically, we sought

to identify enhancers and suppressors of two Notch-related phenotypes generated by

overexpression of wild type (WT) Drosophila Psn in third instar larval wing discs using the

GAL4-UAS system. For this purpose, a pannier-GAL4 (pnr-GAL4) line was used to drive

expression of Psn in the thoracic region of the wing imaginal disc and a cut-GAL4 line was used

to promote expression of Psn along the presumptive wing margin. Psn overexpression resulted

in supernumerary macrochaetes on the scutellum (Fig. 2.1 B, arrows point to additional bristles)

and a notched wing phenotype similar to that observed in Notch loss-of- function mutants (Fig.

2.1 F, arrowhead marks notching of the distal wing blade), as compared to controls (Fig. 2.1 A,

arrow points to a WT bristle and Fig. 2.1 E, arrowhead marks a smooth wing margin). The

observation that overexpression of Psn resembles Notch loss-of-function phenotypes has been

previously described and attributed to possible dominant-negative effects [263]. While the

precise mechanisms leading to these phenotypes are presently unknown, the accumulation of

non-functional complexes within the endoplasmic reticulum and Golgi compartments may

decrease endogenous Psn function and, as a result, affect Notch signalling [263]. Both

phenotypes were 80% penetrant at 29°C and were used to screen a collection of P- and EP-

insertion alleles for those that could enhance or suppress the phenotypes (representative

examples are shown in Fig. 2.1 C, D, G and H). Modification of both Psn-dependent

phenotypes confirmed a genetic interaction.

2.4.2 A Screen to identify Presenilin modifiers

In our genetic modifier screen, we analyzed approximately 1600 P- and EP-element

insertion lines for Psn- interacting genes. Although this is a moderate number of genes

compared to other forward genetic screens [15, 259, 264, 265], or to the number of available

stocks [266], we obtained 178 genes that interacted with at least one of the Psn overexpression

34

Figure 2.1 Suppression or enhancement of Psn-dependent Notch-related phenotypes in adult nota and wings. (A, E) WT flies have four scutellar macrochaetae (arrow points to one) and a smooth distal wing margin (arrowhead). (B, F) Overexpression of Psn in imaginal discs results in supernumerary scutellar bristles (arrows) and a notched distal wing blade (arrowhead). (C, G) Representative suppression of Psn-dependent phenotypes in the notum and wing by the hypomorphic allele, FKBP14EP2019. (D, H) Enhancement of Psn-dependent phenotypes involves tufting of bristles on the scutum and scutellum and multiple notches along the distal wing blade.

recombinant lines. We screened 105 (59%) of the modifiers a second time to validate the

genetic interaction. The candidate genes were categorized according to their predicted

molecular function (Table 2.1), as defined in the FB2008_07 version of the Drosophila

melanogaster genome database [57]. Identification of known factors involved in Notch

signalling, which relies on Psn-dependent γ-secretase activity, included α-Adaptin, bft (bereft),

Bre1, CycE (Cyclin E), emc (extra macrochaetae), Eps-15, esg (escargot), kis (kismet), L

(Lobe), l(2)44DEa, mam (mastermind), Nle (Notchless), numb, osa, ptc (patched), Rab5 (Rab-

protein 5), Rab6 (Rab-protein 6), Ras85D (Ras oncogene at 85D), and tkv (thickveins) [97, 259,

267-278], and factors that associate with PSEN in vertebrates, including the calcium release

channel Ryanodine receptor, further validates the results from our screen. Another modifier

known as thread (th), which encodes an anti-apoptotic factor [279], also suppressed Psn

overexpression phenotypes in the eye [263]. Therefore, we obtained approximately 1 in 8 genes

known to be involved in Psn function, suggesting that other candidates may also be involved in

Psn activity. Interestingly, a subset of modifiers from our screen overlapped with factors

identified in a screen for novel Notch pathway members, including achi (achintya), CycE, dac

(dachshund), emc, esg, for (foraging), grh (grainy head), Gsc (Goosecoid), kis, Kr-h1 (Kruppel

homolog 1), lilli (lilliputian), mam, miple2, numb, osa, Ras85D, Sin3A, Sdc (Syndecan), skd

(skuld) and th [259], which increases our confidence in the results from the screen. We

35

identified numerous calcium regulators, in addition to genes required for apoptosis and the stress

response. Genes involved in protein translation, signal transduction and transcription factor

activities were also isolated. Modifiers with specific or unknown functions were categorized as

'other genes'. Here, I describe the modifiers according to their predicted molecular roles.

2.4.2.1 Calcium signalling components

Recent studies have shown that presenilins form low-conductance divalent-cation-

permeable ion channels in planar lipid bilayers, and functions as a passive ER calcium leak

channel [150, 280]. These data are particularly relevant to our screen, since we identified the

intracellular calcium signalling components known as Calmodulin (Cam), CBP (sarcoplasmic

calcium-binding protein), FKBP14 (FK506-binding protein 14), pain (painless) and Ryanodine

receptor (Rya-r44F) [57, 281] as Psn modifiers. Cam interacts with many binding targets,

including the Ryanodine receptor, which is involved in intracellular calcium release in skeletal

and cardiac muscle and neurons [191, 282-285]. We found that the immunophilin FKBP14

plays a role in Psn protein maintenance in Drosophila (described in Chapter 3). In our genetic

screen, a Cam hypomorph, Cam11356, suppressed both Psn-dependent phenotypes, and a Cam6806

null line suppressed wing notching phenotypes (Table 2.1). Moreover, the Cam10379

hypormorph suppressed bristle phenotypes in the notum, but did not affect wing notching

phenotypes, suggesting that this insertion may allow for more gene expression compared to the

null allele. Surprisingly, the Cam10491 hypomorph exhibited enhanced wing notching (Table

2.1), indicating that this insertion may affect gene expression of nearby genes. We also

observed that an FKBP14 hypomorph, known as FKBP14EP2019, suppressed notum bristle

phenotypes, but did not affect wing notching defects (Table 2.1). We subsequently performed

an excision screen using this allele (described in Chapter 3) and obtained an FKBP14 null allele,

FKBP14D58, that suppressed both phenotypes and an FKBP14 revertant, referred to as

FKBP14D34, that did not affect either phenotype (Table 2.1). This confirmed the genetic

interaction between Psn and FKBP14. The FKBP14EP2206 and FKBP1410962 insertions

suppressed only one of the phenotypes (Table 2.1), and FKBP1410962 also enhanced notum

bristle phenotypes, suggesting that this insertion may disrupt expression of adjacent genes. We

also found that Rya-r44F10559 suppressed both phenotypes, and both CBPEP1523 and painEP2621

suppressed Psn-dependent notum phenotypes (Table 2.1). Therefore, these data provide strong

evidence that Presenilin function is integral to intracellular calcium sensing and release.

36

2.4.2.2 Regulators of stress response and protein folding

Genes that encode heat shock proteins and chaperones are upregulated in response to

physiological stress stimuli, such as heat shock or accumulation of unfolded proteins in the ER

[205, 208, 286, 287]. We identified the stress response genes copper chaperone for SOD1

(CCS), Hsp70/Hsp90 organizing protein homolog (hop), Heat shock protein 60 related

(Hsp60B), Heat shock protein 83 (Hsp83) and the putative chaperone, FKBP14, as Psn-

interacting genes. Specifically, we isolated CCSEP2511, Hop10483, Hsp60B11772 and Hsp8312064 as

suppressors of Psn-dependent notum or wing phenotypes (Table 2.1). The Hsp60B11772 line also

enhanced wing notching phenotypes, suggesting that Hsp60B activity may be involved during

wing disc development. Since Psn overexpression results in aggregation of uncleaved protein

(personal observation; [263]), identification of these genes may have been in response to global

effects of protein aggregation. Further analysis will elucidate whether these factors are directly

required for endogenous Psn activity, which will aid in our understanding of the role that Psn

may play during the stress response.

2.4.2.3 Cell cycle and apoptotic factors

The etiology of Alzheimer’s disease (AD) involves cell death and formation of amyloid

plaques and neurofibrillary tangles. Apoptosis is a regulated form of programmed cell death,

and plays a significant role in AD-associated neurotoxicity, oxidative stress, disrupted

mitochondrial function and increased intracellular calcium levels [155, 169-171, 288]. We

identified key apoptotic factors and genes involved in cell cycling as Psn modifiers, including

CycE, downstream of receptor kinase (drk), eIF-5A, expanded (ex), for, fray, Hop, held out

wings (how), lilli, Ornithine decarboxylase antizyme (oda aka. guf), par-1, Ras85D, Ribosomal

protein L30 (RpL30 aka. plume), smt3, th, tkv and turtle (tutl) [264, 289-299]. The majority of

these transposons suppressed Psn-dependent phenotypes in the notum or in the wing, while

for12326 enhanced wing notching phenotypes (Table 2.1). Interestingly, drk12378, Hop10483,

lilli10944, lilli10388, oda10414, Ras85D11694, th12093 and tkv10504 suppressed both phenotypes.

However, oda10414 may also affect a nearby gene known as Small ribonucleoprotein Sm D3

(SmD3) [57]. Together, these results indicate a possible role for Psn function in apoptosis and

cell cycling. Examining the effects of apoptotic stimuli in AD-related pathology may therefore

aid in our understanding of the underlying cell death mechanisms in disease.

37

Table 2.1. Enhancers and suppressors of Psn phenotypes

Gene Cytology Line ID Modification Molecular Function

cut-Psn pnr-Psn

Calcium signalling

Cam 48F1 10379 10491 11356 6806

NE EP SP SP

SP ---- SP ----

calcium binding; myosin heavy chain binding

CBP 7B2 EP1523 NE SP calcium binding

FKBP14 57E6 EP2019 EP2206 10962 D58 D34

NE NE SP SP NE

SP SP EP SP NE

calcium binding; FK506 binding; peptidyl prolyl cis/trans isomerase activity

pain 60E5 EP2621 NE SP calcium channel activity

Rya-r44F 44F1-2 10559 SP SP ryanodine-sensitive calcium channel activity

Cell cycle regulation

CycE 35D4 11396 NE SP protein kinase regulator activity

ex 21C2 10630 SP ---- regulation of cell proliferation

Notch signalling

alpha-Adaptin 21C2 12319 NE SP protein transporter activity

bft 33B2-33B3 EP980 ---- SP unknown

Bre1 64E8 11541 SP ---- E3 ubiquitin ligase activity

emc 61C9 11629 ---- SP transcription corepressor activity

Eps-15 60E1 EP2554 NE SP calcium ion binding

mam 50C23-50D3 EP2198 EP ---- unknown

Nle 21E2 11099 NE SP Notch binding

numb 30B3-5 11278 EP SP Notch binding; nucleic acid binding

Post-translation modification

Akap200 29C3-29C4 EP2072 ---- SP protein kinase A binding

alpha-4GT1 23C4 EP2097 NE SP galactosyltransferase activity

CG9619 76A3-76A4 10971 SP ---- protein phosphatase type 1 regulator activity

CG11070 26F3-26F4 EP2597 ---- SP ubiquitin-protein ligase activity

CG11489 79D4 10036 SP ---- serine/threonine kinase activity

CG16936 60E1 EP2620 ---- SP glutathione transferase activity

38

for 24A2-4 12326 EP ---- serine/threonine kinase activity

fray 91B4-5 11721 SP ---- serine/threonine kinase activity

grp 36A10 EP2566 ---- SP serine/threonine kinase activity

key 60E1 11044 SP ---- IkappaB kinase activity

Oda 48E4 10414 SP SP ornithine decarboxylase inhibitor activity

par-1 56D9-11 10615 SP NE serine/threonine kinase activity

park 78C2 10006 SP NE methyltransferase activity, ubiquitin ligase activity

Pka-R2 46D1-46D4 EP2162 SP NE protein kinase activity

RhoGAP71E 71E1-71E2 12100 SP SP inorganic diphosphatase activity

rols 68F1 11729 SP ---- lipase activity

smt3 27C7 10419 SP NE ubiquitin- like activity

Src64B 64B11-64B12 11578 ---- SP tyrosine kinase activity

th 72D1 12093 SP SP ubiquitin ligase activity

tna 67F1 12080 SP SP zinc ion binding

tutl 24E1 10979 SP ---- kinase activity

Protein translation

Aats-val 49F7 10452 ---- SP mRNA binding; tRNA ligase activity

CG2950 25A8-25B1 10231 SP ---- RNA binding

eIF2B-gamma 52A10 10766 EP SP translation initiation factor activity

eIF2B-beta 3B2 10785 SP NE translation initiation factor activity

eIF-5A 60B7 11039 NE SP translation initiation factor activity

Glycogenin 57D1 EP2045 ---- SP glycogenin glucosyltransferase activity

hoip 30C5 10654 NE SP mRNA binding

how 94A1 12151 ---- SP mRNA binding

mRpL51 29D4 11655 ---- EP structural constituent of ribosome

mRpL9 88F1 10204 SP ---- structural constituent of ribosome

Nat1 18C7 10760 SP ---- alpha-N-acetyltransferase activity

RpL30 37B9 10952 SP ---- structural constituent of ribosome

RpS26 36F4 12048 NE SP structural constituent of ribosome

39

Signal transduction

bnl 92B2-3 11704 NE SP fibroblast growth factor receptor binding

drk 50A13 12378 SP SP sevenless binding; SH3/SH2 adaptor activity

EDTP 54B7-54B15 EP922 ---- SP protein tyrosine phosphatase activity

ptc 44D5-E1 10514 SP ---- hedgehog receptor activity

Rab5 22E1 10786 SP SP GTPase activity

Rab6 33C4 10446 SP SP GTPase activity

Ras85D 85D19 11694 SP SP GTPase activity

rdgB 12C1-12C4 EP1534 EP1539 EP1648

NE NE ----

SP SP SP

phosphatidylcholine transmembrane transporter activity

Rhp 13E18 EP1647 NE SP GTP-Rho binding

Sdc 57E1-57E6 10431 12377

SP NE

SP SP

transmembrane receptor activity

Spt-1 49F4 EP873 NE SP serine C-palmitoyltransferase activity

tkv 25D1-2 10504 SP SP transforming growth factor beta receptor activity, type I; kinase activity

Tl 97D2 10343 SP ---- transmembrane receptor activity, cytokine binding

w 3B6 10797 SP ---- transmembrane receptor activity, eye pigment precursor transporter activity

Stress response

CCS 46F1 EP2511 NE SP superoxide dismutase Cu chaperone activity

Hop 21B8 10483 SP SP unfolded protein binding

Hsp60B 21E2 11772 EP SP unfolded protein binding

Hsp83 63B11 12064 NE SP unfolded protein binding

l(3)01239 68A4 11526 NE EP chaperone binding, unfolded protein binding

Transcription factors

achi 49A10 EP2107 NE SP transcription corepressor activity

bel 85A5 10063 SP EP RNA helicase activity

btsz 88D4-88D5 11743 NE EP DNA binding

CG15835 43F2 EP2055 ---- SP transcription repressor activity

CrebA 71E1 10183 SP ---- RNA polymerase II activity

40

dac 36A1-2 12047 NE SP RNA polymerase II activity

Dek 53D14 EP2523 EP ---- nucleic acid binding

esg 35D2 10359 SP ---- RNA polymerase II transcription factor activity

gcm 30B12 5445 NE SP transcription factor activity

Gef26 26C3 11102 NE SP guanyl-nucleotide exchange factor activity

grh 54E10-F1 10460 SP NE RNA polymerase II activity

Gsc 21E2 11404 SP SP transcription factor activity

His2Av 97D3 11650 NE EP DNA binding

kis 21B4-21B5 11023 SP SP ATP-dependent helicase activity

Kr-h1 26B5 10381 SP ---- transcription factor activity

lid 26B2 10403 SP SP transcription regulator activity

lilli 23C1-3 10944 10388 EP2172

SP SP SP

SP SP ----

transcription factor activity

Mcm2 84F6 12122 SP SP DNA helicase activity

melt 65E4-65E5 EP3110 NE SP phosphoinositide binding, transcriptional repressor activity

mus209 56F11 10361 NE SP DNA polymerase processivity factor activity

noc 35B2 EP2173 SP NE RNA polymerase II activity

NTPase 23C1 EP2172 SP ---- nucleoside-diphosphatase activity

NUCB1 75A2 10581 SP NE DNA binding; calcium ion binding

osa 90C1-2 11486 SP EP transcription coactivator activity

pcm 18C7 EP1526 NE SP exoribonuclease activity

retn 59F5 11200 SP ---- transcription factor activity

simj 67E6 11548 NE SP transcription repressor activity

Sin3A 49B5-49B7 EP2580 NE SP RNA polymerase II transcription factor activity

skd 78A2 10198 SP SP RNA polymerase II transcription mediator activity

step 39F3-40A1 EP2195 NE SP ARF guanyl-nucleotide exchange factor activity

zfh1 (with osp) 100A4-5 11515 NE EP RNA polymerase II activity

41

Other genes

Acer 29D4 10679 SP ---- peptidyl-dipeptidase A activity

Ance-5 60E5 EP2205 EP NE peptidyl-dipeptidase A activity

Arp87C 87C5 11424 SP ---- structural constituent of cytoskeleton

boc unmapped 11760 SP ---- unknown

BRWD3 95F12-95F13 12155 NE SP unknown

Bsg 28E3-28E5 11096 NE SP unknown

cathD 43E18 EP2151 SP ---- cathepsin D activity

Cg25C 25C1 11110 SP ---- extracellular matrix constituent

CG1702 19D1 EP1525 NE SP glutathione transferase activity

CG2811 60E5 EP2135 SP NE unknown

CG3074 58C7-58D1 EP2059 ---- SP cathepsin B activity

CG3558 23C4 EP2122 SP NE unknown

CG5149 27F3 10225 EP ---- structural molecule activity

CG6043 34B3-34B4 11227 SP ---- unknown

CG10433 57F3 EP2516 ---- SP unknown

CG11034 25F5 EP2131 SP ---- dipeptidyl-peptidase IV activity

CG11329 26F6 10480 SP SP unknown

CG11885 21C2 EP2211 SP ---- unknown

CG11892 96C9 10049 SP ---- unknown

CG13053 72D10 10185 SP ---- unknown

CG14959 63C2-63C3 EP3041 NE SP chitin binding

CG15432 24F2 10561 SP SP unknown

CG30118 55C9 EP2117 NE SP unknown

CG32206 76A6-76B2 10365 EP NE unknown

Cyp4s3 13A12 10545 SP ---- electron carrier activity

Dlc90F 91A2 11646 NE SP dynein intermediate chain binding

dyl 64B1 10945 SP ---- structural constituent of chitin-based cuticle

fs(3)neo27 89D-89E 10061 SP ---- unknown

L 51A4 EP2209 NE SP unknown

LanB1 28C4-28D1 EP2178 SP ---- unknown

l(2)44DEa 44E1-2 10433 SP ---- fatty-acid-CoA ligase activity

l(2)03832a 39F1 11352 EP EP unknown

l(2)06708 24C7-24C9 12320 SP SP unknown

l(2)k03002a 41C 10355 SP ---- unknown

l(2)k03003 49B1-49B2 10521 SP ---- unknown

l(2)k06502 25F3-4 10626 NE SP unknown

l(2)k08504 44A1-44A2 10790 SP SP unknown

42

l(2)k09022 27C1-27C2 10852 SP NE binding

l(2)k09610 21C6-21C7 10906 ---- SP unknown

l(2)k10239 38A7-38A8 10986 SP ---- unknown

l(2)k11206 25C5-25C6 11017 SP SP unknown

l(3)00534 78D1-78D2 11500 SP SP unknown

l(3)00643 90E1-90E2 11505 NE EP unknown

l(3)10585 92B3-92B5 11750 SP NE unknown

l(3)B12-3-31 60A-60D 10703 SP NE unknown

l(3)j2D3 68F3 12084 SP SP unknown

l(3)j4B9 82E4-82E5 12115 EP SP unknown

l(3)j4E6 75E3-75E5 12105 NE SP unknown

l(3)j5A6 91F11 10308 SP ---- unknown

l(3)j8B9 99F8-99F9 10349 SP ---- unknown

l(3)j11B2 74D3-74D5 12101 SP NE unknown

l(3)j13B3 75C5-75C6 12104 NE SP unknown

l(3)L3130 61C7-61C8 10155 EP ---- unknown

l(3)neo35 84D14 10272 SP ---- unknown

l(3)neo39 87D1-D12 10276 ---- SP unknown

l(3)neo51 92A 10288 SP ---- unknown

l(3)rG166 64A4-64A5 12065 SP NE unknown

l(3)rN346 89A1-A2 10297 SP ---- unknown

l(3)ry16 84A5-84A6 10599 SP NE unknown

l(3)ry141 92F 10634 SP ---- unknown

l(3)s2976 98E1-98E2 12160 NE SP unknown

mex1 71D4 11532 NE EP unknown

miple2 61B3 11605 SP EP growth factor activity

Mmp2 45F6-46A1 10358 ---- SP metalloendopeptidase activity

ms(3)neo94 77B-77C 10076 SP NE unknown

mtacp1 61F6 12061 SP SP NADH dehydrogenase activity

nimC3 34F1 10487 NE SP unknown

osp (with zfh1) 35B3-35B4 11515 NE EP unknown

Reg-5 60E1 EP1065 ---- SP unknown

Rpt1 43E7 10437 SP SP endopeptidase activity

scat 30B3 11767 NE SP transmembrane transporter activity

sec31 44F3 10915 SP ---- unknown

Thiolase 60A5 10420 SP SP acetyl-CoA C-acyltransferase activity

unch unmapped 11142 SP ---- unknown

Vap-33-1 3F9 10220 SP ---- structural molecule activity

Vha55 87C2-3 12128 NE SP hydrogen-exporting ATPase activity

43

vlc 41F8 12331 ---- SP unknown

ZIP1 42C6 EP2573 SP NE metal ion transmembrane transporter activity

26-29-p 70C10 10178 SP ---- cathepsin K activity

Table 2.1 Molecular Classification of Presenilin Modifiers. Mutations in 178 genes suppressed or enhanced Psn-dependent overexpression phenotypes in the wing (cut-GAL4) and notum (pnr-GAL4). These genes have been categorized according to their predicted molecular functions. Legend: EP (enhanced phenotype), NE (no effect), SP (suppressed phenotype) and ---- (not tested).

2.4.2.4 Signal transduction and trafficking components

Components of various signal transduction pathways were identified in our modifier

screen. These included members of the Epidermal growth factor receptor (EGFR) and

downstream Ras/MAPK signalling pathways such as drk, Gef26, Ras85D and smt3, two

Fibroblast growth factor (FGF) components known as branchless (bnl) and grh, a receptor for

the morphogen Hedgehog (Hh) known as ptc, members of the Notch signalling pathway

including α-Adaptin, bft, Bre1, CycE, emc, Eps-15, esg, kis, L, l(2)44DEa, mam, Nle, numb, ptc,

Rab5, Rab6 and Ras85D, a Transforming Growth Factor Beta/Decapentaplegic (TGFβ/Dpp)

receptor ortholog known as tkv and members of the Wingless and Int-1-related/Wingless

(Wnt/Wg) signalling pathway referred to as osa and par-1 [259, 270, 271, 273, 275, 296, 300-

309]. Furthermore, we isolated factors involved in multiple signalling pathways, including the

cell cycle regulator ex that is involved in EGFR, Notch, TGFβ/Dpp, Hippo and Wnt/Wg

signalling in imaginal epithelia [293, 299, 310], and the transcription factor lilli, which is

involved in EGFR, Ras/MAPK, TGFβ/Dpp and Wnt/Wg signalling during development [311-

313]. Mutations in many of these genes, including α-Adaptin12319, bftEP980, bnl11704, Bre111541,

emc11629, Gef2611102, grh10460, Eps-15EP2554, esg10359, LEP2209, l(2)44DEa10433, Nle11099, par-110615

and ptc10514 suppressed Psn-dependent notum or wing phenotypes, whereas kis11023, Rab510786,

Rab610446, Ras85D11694 and tkv10504 suppressed both phenotypes (Table 2.1). Intriguingly,

mamEP2198 enhanced wing notching phenotypes, whereas numb11278 enhanced wing notching

defects but suppressed bristle notum phenotypes, and osa11486 suppressed wing notching

phenotypes and enhanced notum bristle phenotypes (Table 2.1). Since mam, numb and osa are

known components of the Notch pathway [259], and Notch signalling is involved in lateral

inhibition and inductive signalling during larval wing disc development [118, 314-316], these

effects may be due to different signalling requirements during larval disc development.

44

2.4.2.5 Hematopoietic factors

Hemocytes are specialized Drosophila immune cells involved in phagocytosis

(plasmatocytes), melanization (crystal cell) and encapsulation of parasites (lamellocytes) [139,

142]. Numerous factors, notably the transactivator glial cells missing (gcm) and members of the

Notch, Ras/MAPK, TGFβ/Dpp and Toll signalling pathways are essential for hemocyte

production, proliferation and specialization [138, 140, 142, 317-320]. In addition to gcm, we

identified components of the Notch pathway, Rab5 and skuld (skd) [259], a receptor for the

TGFβ/Dpp pathway, tkv [321], a key component of the Ras/MAPK pathway known as Ras85D

[319] and the Toll (Tl) receptor as Psn- interacting genes. Skuld functions as a transcriptional

coactivator that is required to maintain differences in cell affinities between dorsal and ventral

cells at the presumptive wing margin, and may be required for expression of some Wingless and

Notch target genes [259, 322-324]. We also recovered the serine/threonine kinase for [325], a

transcription co-repressor known as Sin3A [326] and a SUMO-1 ortholog involved in Toll

signalling known as smt3 [327], which were previously identified in a screen for factors

involved in embryonic crystal cell development [320]. We found that gcm5445 and Sin3AEP2580

suppressed notum bristle phenotypes, smt310419 and Tl10343 suppressed wing notching defects and

skd10198 suppressed both phenotypes. Further analysis of the interactions between these factors

and Presenilin may help elucidate the precise role of Presenilin during hematopoiesis.

2.4.2.6 Alzheimer’s disease-associated factors

A link between AD and human angiotensin converting enzyme (ACE), a metalloprotease

of the renin-angiotensin system that regulates blood pressure [328], has been observed in

numerous human genetic studies [329-331]. Common polymorphisms in human ACE associate

with APO-E, a known causative factor for late-onset AD [329-331]. ACE-inhibitors have been

shown to alleviate some of the symptoms of cognitive impairment, although they have also been

shown to increase Aβ levels in vivo [329, 332]. We identified two ACE-like factors in

Drosophila that share enzymatic similarities with human ACE, known as Acer and Ance-5

[333], as putative regulators of Psn function. Acer10679 suppressed wing notching phenotypes

and Ance-5EP2205 enhanced wing notching phenotypes (Table 2.1).

45

2.4.3 Confirmation of the genetic interactions between Presenilin and components of

intracellular calcium signalling

Presenilin is known to interact with numerous components of the intracellular calcium

signalling machinery, including Sorcin, Ryanodine receptor and calsenilin, and to mediate

intracellular calcium levels [148-150, 334-336]. FAD-linked mutations in PSEN1 increase

levels of Ryanodine receptor and calcium release in mammalian cells [167]. Interestingly, we

identified a number of calcium signalling components in our genetic screen, including Cam

[282] and FKBP14 (described in Chapter 3). To confirm an association between Psn and

calcium signalling, we obtained a Cam null allele and I generated an FKBP14 null allele in order

to test their interaction with Psn. Overexpression of Psn in the presumptive notum region

caused ectopic scutellar bristles (Fig. 2.2 B; arrowhead), compared to controls (Fig. 2.2 A, arrow

points to a WT bristle), and Psn overexpression at the presumptive wing margin resulted in

anterior wing margin notching in cut-GAL4/UAS-Psn transgenic flies (Fig. 2.2 E). We

demonstrate that Cam and FKBP14 null mutants suppressed Psn overexpression phenotypes

(Fig. 2.2 D, F and G), while an FKBP14 revertant (FKBP14D34) did not suppress Psn-dependent

bristle or wing phenotypes (Fig. 2.2 C and G). Together, these data suggest a role for calcium

signalling in Psn function.

2.4.4 Psn overexpression levels are reduced in FKBP14 mutants

FKBP14 encodes a protein isomerase believed to be involved in protein folding [181,

188]. I show that FKBP14 forms a complex with Psn holoprotein, and discuss this in Chapter 3.

Since FKBP14D58 caused suppression of Psn-dependent phenotypes, I wanted to examine the

effects of a loss of FKBP14 on Psn protein levels. To do this, I analyzed levels of Psn protein in

FKBP14D58; pnr-GAL4/UAS-Psn-Myc (FKBP14D58; pnr-PM5) and FKBP14D34; pnr-

GAL4/UAS-Psn-Myc (FKBP14D34; pnr-PM5) lines, using an anti-Psn antibody that detects Psn

N-terminal fragments as well as full- length protein [56]. Quantitation of Psn protein levels from

three individual experiments demonstrated up to 25% decrease in Psn N-terminal fragment

(PsnNTF) levels in extracts of FKBP14D58; pnr-PM5 20 h pupae, as compared to FKBP14D34;

pnr-PM5 controls (Fig. 2.3 A and B). These results are statistically significant (p < 0.05; n = 3).

The effects on Psn holoprotein are less consistent, where I observed reduced (Fig. 2.3 A) or

enhanced (data not shown) holoprotein levels in extracts of FKBP14D58; pnr-PM5 flies, as

46

Figure 2.2 Cam and FKBP14 null mutants suppress Psn overexpression phenotypes. (A) WT flies have four scutellar bristles (arrow points to one). Bar, 100 uM. (B) Supernumerary bristles (arrowhead points to one) are observed in pannier-GAL4/UAS-Psn (pnr-Psn) recombinant flies. (C) Psn-dependent bristle phenotypes are unaltered in FKBP14D34; pnr-Psn and (D) are suppressed in FKBP14D58; pnr-Psn

flies. (E) Psn overexpression by cut-GAL4 results in notching along the anterior wing margin. (F) Cam null mutants (Cam6806) suppress cut-GAL4/UAS-Psn (cut-Psn) notching phenotypes. (G) Quantitation data showing suppression of cut-Psn phenotypes by FKBP14 null (FKBP14D58), Cam hypomorphic (Cam11356) and Cam6806 null alleles, compared to controls.

compared to controls. This may be attributable to GAL4/UAS overexpression conditions. More

importantly, since PsnNTF levels are reduced in FKBP14D58; pnr-PM5 pupae, this suggests that

FKBP14 function is required for Psn protein regulation. In agreement with this model, I found

that FKBP14 maintained endogenous PsnNTF levels, as discussed in Chapter 3. Modification of

Psn phenotypes may therefore in some instances be due to specific effects on Psn protein levels.

47

Figure 2.3 Psn protein levels are mildly reduced in FKBP14D58 heterozygotes. (A) A representative blot illustrating levels of Psn protein from extracts of 20 h pupae that were raised at 29°C. Psn protein was detected using anti-PsnNTF antibodies as described in Materials and Methods. Overexpression of Myc-tagged Psn full-length protein directed by pnr-GAL4 results in holoprotein (PsnFL) and increased levels of N-terminal fragments (PsnNTF) in FKBP14D34; pnr-PM5 flies, compared to pnr-GAL4 (pnr), UAS-Psn-Myc (PM5), FKBP14D34; pnr-GAL4 (FKBP14D34; pnr) and FKBP14D58; pnr-GAL4 (FKBP14D58; pnr) controls. A mild reduction in PsnNTF levels is observed in FKBP14D58; pnr-PM5 flies compared to FKBP14D34; pnr-PM5 controls, and is consistent in three independent experiments. FKBP14 protein levels are reduced in FKBP14D58 mutant heterozygotes, compared to controls. Actin was used as a loading control. (B) Quantitation of PsnNTF levels was performed by scanning densitometry of immunoblots using a Fluorchem 8000 Gel Documentation System with Alpha Innotech software. Experiments were repeated independently three times. Values shown correspond to the average intensity of the PsnNTF signal, designated as a percentage of an Integrated Density Value (IDV). Error bars represent standard error of the mean. In FKBP14D58; pnr-PM5 20 h pupae, PsnNTF protein levels (15.6 ± 1.3 % IDV) are reduced compared to FKBP14D34; pnr-PM5 flies (21.0 ± 1.9 % IDV), and this is statistically significant: *p=0.02. Other controls include pnr-GAL4 (pnr), UAS-Psn-Myc (PM5), FKBP14D34; pnr-GAL4 (FKBP14D34; pnr) and FKBP14D58; pnr-GAL4 (FKBP14D58; pnr).

2.5 Discussion

We performed a genetic modifier screen to identify Presenilin- interacting genes in

Drosophila. Our screen was designed to identify enhancers or suppressors of Psn-dependent

phenotypes in the wing and notum. We identified 178 modifiers as putative regulators of

Presenilin function, and 105 of these modifiers were screened twice using two independent Psn-

overexpression lines to confirm an interaction with Psn. Components of the Notch signalling

pathway, which relies on presenilin γ-secretase activity, and two known presenilin- interacting

48

genes, known as Ryanodine receptor and thread, were identified. Therefore, we believe that

this validates the outcome of our screen. Since we recovered 31 modifiers that function as

transcription factors, this suggests that the effects on Psn overexpression may occur at the level

of transcription. Alternatively, these factors may represent non-specific interactions that affect,

for example, GAL4 transgene expression, which has been previously observed in other genetic

modifier screens [259]. One of the modifiers that was identified is an immunophilin known as

FKBP14, and FKBP14 orthologs in yeast were shown to exhibit chaperone activity [209]. My

analysis has shown that PsnNTF levels are reduced in flies that overexpress Psn in an FKBP14

mutant background, suggesting that suppression of Psn overexpression phenotypes by FKBP14

mutants may be due to loss of FKBP14-mediated maintenance of Psn protein levels in vivo.

We found that nearly all of the modifiers suppressed Psn-dependent phenotypes in the

notum and wing. Examination of the interactions between Psn and the candidate genes will

determine their possible roles in AD, and in Psn functions that do not rely on γ-secretase

activity, such as apoptosis regulation [154] and calcium homeostasis [150] within the ER. A

limitation with the design of our screen is the possibility of second-site modifications. RT-PCR

analysis of the surrounding loci will determine which gene is affected by each insertion.

Similarly, obtaining additional null alleles and examining their effects on the Psn phenotypes

will confirm an interaction. Another caveat to a loss-of-function screen is that genes that exhibit

functional redundancy among related genes, or genes that do not exhibit loss-of- function

phenotypes, may not be recovered. However, we were able to identify 178 genes that are

putatively involved in Psn activity.

Presenilin function is complex, with well-characterized roles in γ-secretase proteolyis of

Notch and APP [37, 84], and in calcium regulation, β-catenin destabilization and spectrin

cytoskeletal organization in ovarian follicle cells [91, 150, 337]. Our goal was to identify

additional Psn- interacting genes. Of note, we identified five genes that encode calcium

signalling factors, Cam, CBP, FKBP14, pain and Ryanodine receptor, implicating a role for Psn

function in calcium signalling during development and disease. Interactions between presenilin

and many known calcium regulators, including calsenilin, Sorcin and Ryanodine receptor, are

believed to mediate calcium regulation within the cell [145-149]. Presenilin holoprotein also

forms a passive ER calcium leak channel [150], and disrupted intracellular calcium levels have

been linked consistently to numerous clinical familial Alzheimer’s disease (FAD) mutations in

human presenilins [151-153]. Importantly, defects in calcium homeostasis alter proteolytic

processing of APP, a key factor contributing to the appearance of amyloid (Aβ) plaques in

49

brains of patients with Alzheimer’s disease [22]. Alterations in Ryanodine receptor have also

been linked to FAD neurofibrillary and Aβ neurotoxicity [167, 338], highlighting a tight link

between calcium homeostasis and AD. These effects may be mediated through protein-protein

interactions between presenilin and calcium signalling factors. For instance, presenilin-1 co-

immunoprecipitated with Ryanodine receptor and mutations in presenilin-1 increased Ryanodine

receptor protein levels and enhanced intracellular calcium release [167, 334, 335]. Elevated

intracellular calcium levels have also been shown to enhance the interaction between presenilin-

2 and Sorcin, a known modulator of Ryanodine receptor [149]. Together, these data strongly

suggest a role for presenilin in intracellular calcium signalling.

Presenilins also modulate aspects of intracellular apoptosis regulation. Several studies

demonstrate that presenilin C-terminal domains are substrates for caspases, such as caspase-3

[173, 174]. In addition, presenilin-1-deficient neurons exhibited increased levels of apoptosis

and caspase-3 activation leading to neuronal degeneration [176]. FAD-linked presenilin-1

mutations also enhanced the apoptotic effects resulting from oxidative stress and caspase

activation in primary cultured neurons from transgenic murine brains [175, 176]. Similarly,

Psn- induced eye phenotypes in Drosophila can be suppressed by the anti-apoptotic factor, th

[263]. Importantly, presenilin-1 and presenilin-2 have been shown to form multimeric

complexes with members of the Bcl-2 family of proteins, including Bcl-2 and FKBP38, in the

ER/Golgi [154]. Bcl-2 is an anti-apoptotic factor that binds to FKBP38, a mitochondrial-

localized immunophilin, thereby inhibiting intracellular apoptosis [179, 180]. Presenilins target

Bcl-2 and FKBP38 to ER/Golgi compartments promoting their degradation, and rendering the

cell susceptible to apoptosis, independent of γ-secretase activity [154]. FAD mutants enhance

neuronal cell death by targeting more Bcl-2 to ER/Golgi fractions compared to WT presenilin

[154]. Apoptotic stimuli may therefore accelerate AD-related pathology. We identified

mutations in the apoptotic factors, ex and th, and the cell proliferation gene CycE, which further

implicates Psn function in apoptosis and cell cycle maintenance. The membrane-associated

cytoplasmic protein Ex, a member of the FERM family of proteins, acts as an upstream

regulator of the Hippo signalling pathway, which co-ordinately restricts cell and tissue growth

through regulation of th and CycE transcription [288, 291, 310, 339]. In turn, Hippo signalling

inhibits ex transcriptional levels, creating a negative feedback loop for regulation of the pathway

[339]. Since calcium has also been implicated in neuronal viability and apoptosis, examining

the effects of calcium and apoptosis misregulation in disease has become a significant part of

current AD research. Identification of factors involved in calcium homeostasis and apoptosis

regulation as putative Psn- interacting genes will be able to contribute to these efforts.

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Psn overexpression caused accumulation of holoprotein, and this is believed to result in

dominant-negative phenotypes [263]. Mutations in genes involved in protein folding and

chaperone activity were identified as Psn-interacting genes, including CCS, Hop, Hsp60B,

Hsp83 and FKBP14. Heat shock proteins promote proper protein folding by preventing protein

aggregation, and the co-chaperone Hop coordinates the protein folding activities of Hsp70 and

Hsp90 in vitro [340]. CCS orthologs in humans and yeast exhibit copper chaperone activity,

delivering copper to an antioxidant enzyme known as Cu,Zn superoxide dismutase (SOD1) and

mediating activation of SOD1 in response to oxidative stress [341, 342]. Drosophila CCS is

highly conserved with human CCS [343], suggesting that fly CCS may be functionally

conserved. In flies, Hsp60B is essential for male fertility [344], and Hsp83 is required for Raf-

mediated signalling during eye development, involving a serine/threonine kinase cascade that

leads to MAPK translocation to the nucleus and subsequent gene expression [345]. FKBP14

orthologs in yeast are involved in peptidyl-prolyl cis-trans isomerase activity and chaperone

activity [209], and I have demonstrated an involvement of Drosophila FKBP14 in Psn protein

stability and Notch-related peripheral nervous system development, as described in Chapter 3.

Thus, these data suggest that protein folding factors may be involved in facilitating Psn protein

stability. Further analysis of mutant alleles will aid in our understanding of the role that these

factors may play in Psn protein biology.

Numerous studies have shown that convergence of the EGFR, FGF, TGFβ/Dpp, Wnt/Wg

and Notch signalling pathways is highly regulated, for instance, during cell fate specification of

developing embryos [346-349]. An interplay between the FGF and Notch signalling pathways

was also shown to mediate cell migration and branching cell fate decisions during tracheal

tubulogenesis [350]. In particular, the involvement of EGFR, Hh, Notch, TGFβ/Dpp and

Wnt/Wg signalling during wing disc development [301, 351, 352] may account for the number

of factors from these pathways that were identified in our genetic screen. We recovered the

FGF signalling components, bnl and grh, EGFR and Ras/MAPK factors known as drk, Gef26,

Ras85D and smt3, the Hh receptor ptc, components of the Notch pathway, including α-Adaptin,

bft, Bre1, CycE, emc, Eps-15, esg, kis, L, l(2)44DEa, mam, Nle, numb, ptc, Rab5, Rab6 and

Ras85D, the Dpp receptor tkv and the Wnt/Wg pathway members osa and par-1 [259, 271, 273,

275, 296, 300-309] as Psn- interacting genes. Osa has been shown to associate with the Brahma

chromatin remodelling complex to repress Wingless target genes and the achaete-scute

proneural genes [300, 305, 324]. Loss of osa function suppressed Psn-dependent wing

phenotypes and enhanced Psn-dependent bristle defects, suggesting that osa may play multiple

roles in Psn activity. Consistent with this model, osa has been shown to mediate activation or

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repression of target gene expression of the LIM-homeodomain protein Apterous during

establishment of the dorsal-ventral boundary of the wing [300]. Another transcription factor

identified in our screen, lilli, genetically interacts with members of multiple signal transduction

pathways, and may represent a pair-rule gene involved in embryo segmentation and

cytoskeleton regulation [311]. Lilli may also be involved in Notch signalling [259]. Together,

these data implicate Psn function in multiple signalling paradigms during development.

Endocytosis and endosomal sorting of Notch and its ligands by clathrin coat components,

shibire (the Drosophila ortholog of dynamin), liquid facets (the Drosophila ortholog of epsin),

the Rab proteins Rab5, Rab7, Rab11 and E3 ubiquitin ligases such as neuralized are important

for developmental regulation of the Notch pathway [353-357]. Internalization of the Notch

receptor bound by its ligands in the signal-sending cell catalyzes Notch cleavage by the

presenilin-dependent g-secretase complex in the signal-receiving cell [97]. This releases the

Notch intracellular domain that translocates to the nucleus and associates with Suppressor of

Hairless (Su[H]) and other factors to activate target gene expression [354, 356, 358, 359]. We

recovered the small GTPases Rab5 and Rab6, the Notch antagonist numb and a component of

the endocytic machinery known as a-Adaptin [275] as putative regulators of Psn function. In

addition to its role in signal transduction, Rab5 has been implicated in clathrin-coated vesicle

formation and fusion between early endosomes, and may be involved in sorting of Notch to

recycling endosomes and late endosomes [273, 354, 356]. Rab5 has also been implicated in Aβ

peptide regulation, since Rab5 overexpression in mammalian cells enhanced levels of β-

secretase cleaved APP C-terminal fragments, and Aβ40 and Aβ42 levels [360]. Rab6 is

involved in retrograde Golgi-ER transport and Drosophila Rab6 genetically interacted with

Notch and may be required for Notch transport through the Golgi [278, 361]. Recently, numb

has been shown to associate with the clathrin coat component, a-Adaptin, inhibiting Notch

signalling in the signal-sending cell [275]. Thus, intracellular trafficking may play a role in Psn

activity and further examination of the association between Psn and intracellular trafficking

factors will help elucidate the roles of Psn and Notch during development.

During embryogenesis, hemocytes are involved in the production of extracellular matrix

components of basement membranes that surround muscle and fat cells and deposit along the

ventral nerve cord [362, 363]. Hemocytes are also involved in adult wing maturation

immediately following eclosion [364]. In vertebrates, the TGFβ signalling pathway regulates

many stages of hematopoiesis [365] and the orthologous Dpp pathway in Drosophila plays an

indirect role in crystal cell development [320]. We identified the TGFβ/Dpp pathway receptor

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tkv and other factors involved in embryonic crystal cell development, such as for and smt3 [320,

325, 327], as Psn- interacting genes. The Ras/MAPK pathway may also be involved in

hemocyte proliferation [319], and we identified a key component known as Ras85D as a Psn

modifier. Moreover, hemocyte differentiation is mediated by the transactivator gcm, the Toll

pathway and members of the Notch pathway [138, 140, 142, 318-320], and we identified

mutations in gcm, Tl and Notch signalling components in our genetic screen. I also observed

hemocyte-specific localization of FKBP14 in embryos, as described in Chapter 3, indicating that

FKBP14 function is involved during embryonic hematopoiesis. Intriguingly, another member of

the FKBP family of immunophilins, known as FKBP59, has been implicated in crystal cell and

lymph gland development [320]. Together, these data are compelling evidence for a role of Psn

in Drosophila hematopoiesis. Similarly, presenilins have been identified in both lymphoid and

myeloid blood cell development in vertebrates [366]. Further analysis will therefore aid our

understanding of the precise role of presenilin during blood cell development.

A number of genes that were identified in our screen correspond with genes that were

recovered in other developmental screens [15, 259, 264, 301, 319, 320, 367]. As mentioned, we

identified 22 modifiers that were also recovered in a screen for Notch pathway genetic

interactors [259]. Other factors, such as drk, mam, osa, ptc, Ras85D, and tkv were isolated in

our screen and in a screen for regulators of EGFR and TGFβ/Dpp signalling in wing

development [301]. Osa, a member of the Brahma complex, was also identified in a screen for

factors involved in boundary formation in the wing disc, in addition to lilli and skuld [324], and

in Apterous target gene expression in the wing [300]. Delta-Notch signalling may regulate

Brahma complex activity during cell fate specification within the SOP lineage, since Delta was

identified in a screen for enhancers of Brahma-dependent eye and nota phenotypes [368]. CycE

was shown to interact with components of the Brahma complex, in mediating the cell cycle

[369], and CycE and osa were shown to associate with cut activity during wing margin

development [367]. We also isolated two genes that were shown to genetically modify Tau-

induced neurodegeneration in Drosophila, known as par-1 and th [15]. These results indicate

that our modifiers may represent genes involved in numerous pathways that are critical for

development. We are thus more confident in the results from our screen, since there was

overlap with previous screens.

A comparison of human disease-associated genes, that have at least one mutant allele in

the Online Mendelian Inheritance in Man (OMIM) database, against the Drosophila genome

sequence identified 77% of 929 known human disease genes that share similarity with 548

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unique fly gene sequences [370]. This information can be accessed on a database known as

Homophila [370, 371]. Key molecular pathways required for development are shared between

humans and Drosophila, and this led to the development of a number of Drosophila models of

human disease. Drosophila models of neurodegeneration exhibit partial replication of

neurodegenerative disease in humans, including Alzheimer’s disease [242-245]. Since several

aspects of Alzheimer’s disease have recently been attributed to defects in the renin-angiotensin

system that regulates blood pressure [328-331], we were intrigued by the identification of two

functional orthologs of the human angiotensin converting enzyme in our genetic screen, known

as Drosophila Acer and Ance-5 [333]. This suggests that Psn function may be involved in

modulating hypertension. Emerging data from genetic screens in model organisms may

therefore aid in our understanding of the molecular basis of human disease.

2.6 Acknowledgements

The cut-GAL4/UAS-Psn and pannier-GAL4/UAS-Psn lines used in the genetic screen were

generated by Dr. Izhar Livne-Bar. James Hughes helped with the genetic screen. The cut-

GAL4/UAS-Psn line used in Fig. 2.2 E – G was created by Kinga Michno.

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CHAPTER 3

THE INTERACTION BETWEEN FKBP14 AND PRESENILIN MEDIATES NOTCH

SIGNALLING DURING DROSOPHILA DEVELOPMENT

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3.1 Summary

FK506-binding proteins (FKBPs) are peptidyl-prolyl cis-trans isomerases that are

considered integral to protein folding, assembly and trafficking. Here I have characterized the

function of FKBP14 in Drosophila. I found that null mutations in FKBP14 are lethal and give

rise to defects in eye, notum and wing development that arise, in part, from reduced expression

of Notch target genes, such as cut, wingless and Enhancer of split. Interestingly, Notch receptor

trafficking is unaffected in FKBP14 mutants, suggesting that FKBP14 may affect Notch

signalling downstream of trafficking to the plasma membrane. Consistent with this, I find that

FKBP14 colocalizes and binds to Presenilin, a key component of the g-secretase complex that

cleaves transmembrane proteins, such as Notch and Amyloid Precursor Protein. I also

demonstrate that Presenilin levels are reduced in FKBP14 mutants and that this occurs post-

transcriptionally. Altogether, my data support a model whereby FKBP14 stabilizes Presenilin

protein, allowing for a functional g-secretase complex that may mediate signal transduction.

3.2 Introduction

FK506-binding proteins (FKBPs) belong to a family of peptidyl-prolyl cis-trans

isomerases that are considered integral to the folding of proteins into their native conformations

[181, 188]. The peptidyl-prolyl cis-trans isomerase (PPIase) domain is characteristic of these

proteins, and encompasses the catalytic protein folding activity [224]. PPIase domains found in

low molecular weight FKBPs, such as those in FKBP12 [372] and FKBP13 [206] are highly

conserved. FKBPs were originally identified as the predominant cytosolic targets of the

immunosuppressant drugs FK506 and Rapamycin [188, 223, 224]. A portion of the FK506-

binding sequence, known as the AYG motif, and other residues in the drug binding pocket, are

found in over 40 FKBPs from six diverse genomes [373], including Drosophila FKBP14.

Binding to these drugs inhibits PPIase activity and affinity for the drugs varies amongst FKBPs

[224]. In addition to the PPIase domain, FKBP14 orthologs from Neurospora crassa contain a

C-terminal motif required for their dimerization [201] and mammalian FKBP23 contains a C-

terminal EF-hand motif and an ER retention sequence [202].

FKBPs have a wide phylogenetic distribution ranging from yeast to humans. Although

FKBPs are involved in a wide variety of biological processes, many exhibit distinct subcellular

localization and appear to bind to specific protein targets [374]. The cytosolic FKBP12 binds

56

and modulates the calcium release channels known as IP3R and Ryanodine receptor [195, 372],

while FKBP13 is upregulated in the endoplasmic reticulum (ER) during the unfolded protein

response [205, 208]. FKBP38 forms a complex with Presenilins and Bcl-2 and promotes

apoptosis in a g-secretase-independent manner [154]. While the function of most FKBPs

appears to depend on the highly conserved PPIase domain, increasing evidence suggests that

FKBPs bind to distinct protein targets and regulate specific cellular functions.

The function of FKBP14 in multicellular organisms is not well understood. Here I show

that Drosophila FKBP14 is required for the development of adult sensory structures in the eye,

notum and wing, and for regulation of apoptosis in larval imaginal discs. The defects in wing

margin specification observed in FKBP14 mutants are associated with reduced expression of

downstream targets of the Notch signalling pathway, whereas notal bristle defects may be

caused by reduced proneural gene expression and Notch signalling activity. Interestingly, Notch

receptor levels at the plasma membrane are unaffected in FKBP14 mutants, implicating normal

intracellular trafficking. Consistent with a role for FKBP14 in the Notch signalling pathway, I

found that FKBP14 genetically interacts with both Notch and Presenilin (Psn), which encodes a

key component of the g-secretase complex that cleaves Notch. In addition, FKBP14 and Psn

colocalize in the ER where they form a complex in vivo. Finally, I show that Psn protein levels,

but not RNA levels, are reduced up to 90% in FKBP14 mutants. Altogether, these data support

a model in which FKBP14 stabilizes Psn protein in the ER, which may allow for g-secretase-

mediated activation of Notch signalling.



The P-element line, w+; FKBP14EP2019/CyO possesses an insertion in the first intron of the

FKBP14 locus. An excision screen was performed on FKBP14EP2019 to generate FKBP14

alleles, as described [266]. The P-element lies 101 bp downstream of the first exon and 1639 bp

upstream of the translational start site within exon 2. Sara lies 58 bp downstream from exon 5

of FKBP14 and CG10496 is 1691 bp upstream of the first exon of FKBP14-RA, as described in

Flybase [57]. FKBP14EP2019 virgin females were crossed to Sp/CyO; Sb, ∆2-3/TM3, Ser males

carrying a source of transposase that mobilizes P-elements. The appearance of variegated eyes

57

in the progeny indicated mobilization of the EP-element. Male progeny exhibiting variegated

eyes were crossed to IF2/SM5, Cy balancer females. Individual male progeny from this cross

that exhibited white eyes, indicating excision of the EP-element, and the Cy marker were

crossed to IF2/SM5, Cy balancer females. Individual sibling male and female progeny

exhibiting white eyes, the Cy marker and the absence of the IF marker, were mated and then

maintained in a w+ background over the balancer CyO, Kruppel-GFP to identify homozygotes.

Genomic DNA from 11 individual lines was analyzed for deletions in exons 1 and 2 of the

FKBP14-RA locus using PCR. 4 lethal lines were identified, one of which exhibited a deletion

in the FKBP14 locus. This imprecise excision line, referred to as FKBP14D58, displayed a 2405

bp deletion, including residues 105-145 of exon 1 and the entire exon 2. Breakpoints were

determined by DNA sequence analysis from ACTG. A precise excision line, FKBP14D34, was

also generated and confirmed by DNA sequence analysis from ACTG. This line is used as a

genetic control.

Psn overexpression and mutant analysis have been described [56]. Sequence alignments

were preformed using ClustalW [375].

3.3.2 Drosophila stocks

The Psn null line, Psnw6rp, has been described [56]. The L Pin/CyO-GFP (5194), and

Df(1)N-8/FM7c (729) lines were obtained from the Bloomington Stock Center. Stock numbers

are indicated in brackets. FKBP14EP2019 is available from the Szeged Stock Center

(P[EP]Fkbp13EP2019). The SP/CyO; Sb, ∆ 2-3/TM3, Ser and L Pin/SM5, Cy lines are maintained

in our lab.

3.3.3 RT-PCR

Total RNA was extracted using Trizol (Invitrogen) and reverse transcribed using the

Superscript First-Strand system (Invitrogen) to prepare cDNA, and performed in triplicate.

Specifically, 4 mg of total RNA was used to synthesize cDNA and 2 mL of this reaction was

subsequently used for PCR amplification of Psn, using the primers,

GGCTGCCATTTCTATTTGGG (+) and CTGACCACTCTTGCGTGAAAC (-). Gapdh

amplification was used as a control.

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3.3.4 Immunoblot Analysis and Immunoprecipitation

Flies were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1%

SDS, 0.02 molar Tris-Cl pH 8.0, 0.137 molar NaCl, 10% glycerol, 2 uM EDTA) supplemented

with Complete EDTA-free protease inhibitors (Roche). Lysates were centrifuged at 10,000 g

for 15 minutes and supernatants were analyzed with rabbit a-Psn-NTF [56], mouse a-b-tubulin

(DSHB; 1:1000) and rat a-FKBP14 (1:2000) generated as follows: a Drosophila FKBP14

cDNA (GH08925, amino acids 298-828; BDGP) was cloned into pGEX-4T-1/His 6C (Novagen)

to produce an FKBP14-GST fusion protein for polyclonal antibody production in rats

(Antibodies Inc., Davis, CA) and preabsorbed on fixed S2 cells. HRP secondary antibodies

were used (Jackson ImmunoResearch; 1:10 000), and ECL (Pierce) was used to visualize

protein signals. All blots were performed in triplicate. Quantitation was performed using a

Fluorchem 8000 Gel Documentation System and Alpha Innotech software (San Leandro, CA).

FKBP14 truncation products were not observed in FKBP14D58 extracts, therefore, I believe that

this line constitutes a protein null.

S2 cells were harvested 40 h post-transfection, lysed in 0.1% Triton-X-100 in PBS

supplemented with protease inhibitors (Roche) and precleared with Protein G beads (Sigma) for

20 mins at 4°C. Co-immunoprecipitation reactions containing cleared lysate and 0.8 ug of anti-

Myc (The Hospital for Sick Children, Monoclonal Antibody Facility) in a final volume of 85 uL

were carried out at 4°C for 30 min, followed by the addition of 25 uL of a 70% slurry of beads

and a final incubation at 4°C for 30 min. Beads were washed in lysis buffer (0.1% Triton-X-100

in PBS; 300 uL) and heated at 50°C for 20 min prior to SDS-PAGE (adapted from [376]).

3.3.5 Immunohistochemistry

Immunostaining of third instar wing discs and CNS was performed using standard

procedures [377-379]. S2 cells were stained as described [380]. The following primary

antibodies were used: mouse a-Ac (1:100), mouse a-Ct (1:100), mouse a-Notch extracellular

domain, EGF repeats #12-20 (C458.2H; 1:100), and mouse a-Wg (1:500) all available from

DSHB, mAb323 (E(spl); gift of Dr. S. Bray; 1:1), rat a-FKBP14 (1:100), mouse a-KDEL

(MBL; 1:100), mouse a-p120 (Calbiochem; 1:500), guinea pig a-Sens (gift of Dr. H. Bellen;

1:1000) and a rabbit a-Psn-CTF antibody generated to a peptide corresponding to amino acids

426-441 (CG18803-PB; NCBI) that was affinity purified (Antibodies Inc., Davis, CA; 1:200).

59

A488 and Cy3 secondary antibodies were used (Jackson ImmunoResearch; 1:1000). DAPI was

used at 1:5000. Samples were mounted in Dako Mounting Medium (DakoCytomation).

Third instar larval CNS and discs were dissected in PBS, incubated in 0.25 mg/mL

acridine orange (Invitrogen) in PBS for 5 minutes at RT, rinsed in PBS and mounted

immediately in PBS prior to fluorescence microscopy.

3.3.6 Microscopy

Images were acquired at RT using either a Zeiss LSM510 META confocal microscope,

40x/1.2 and 100x/1.3 objectives and standard fluorescence filters or a Leica DMRA2

Fluorescence Microscope equipped with a Hamamatsu Orca-ER digital camera and Improvision

Openlab software, 20x/0.5, 40x/1.25-0.75 and 100x/1.4 objectives, brightfield and standard

filters or a Leica DMLB Fluorescence Microscope, 5x/0.12 objective, brightfield and

CoolSNAP software. Micrographs were analyzed using an FEI XL30 Scanning Electron

Microscope equipped with XL Docu software. Images were processed in Photoshop CS and

Illustrator CS.

3.3.7 Cell culture

Psn cDNA (longest isoform; [57]) was amplified from the clone LD23505 (BDGP) and

cloned into pAc5.1/V5-His (Invitrogen) to generate pAc-Psn-Myc. S2 cells were maintained in

Schneider’s media supplemented with 10% fetal bovine serum, transfected with 3 ug of plasmid

DNA using Cellfectin (Invitrogen) and assays were conducted at RT.

3.4 Results

3.4.1 Molecular Characterization of Drosophila FKBP14

Currently there are 8 known FKBPs in Drosophila that share homology with the

archetypal human FKBP12. Sequence analysis of one of these, Drosophila CG9847, reveals

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that it has a PPIase (FKBP_C) domain, an EF-hand motif and an ER retention signal (Fig. 3.1

A). Although it was originally identified as Fkbp13 [381], its closest mammalian ortholog is

FKBP14 (or FKBP22), with 37% overall sequence identity (Fig. 3.1 B). In contrast, mammalian

FKBP13 shares 26% sequence identity with Drosophila CG9847 and 57% sequence identity

with another Drosophila FKBP, known as CG14715 (Figure 3.2). Taken together, this indicates

that Drosophila CG9847 encodes an ortholog of mammalian FKBP14. Hereafter, I will refer to

the protein encoded by Drosophila CG9847 as Drosophila FKBP14.

Figure 3.1 Drosophila FKBP14 shows homology to vertebrate FKBP14. (A) Drosophila FKBP14 contains an N-terminal signal peptide, a PPIase domain (FKBP_C; dark grey), an EF-hand calcium binding motif (EFh; blue) and a C-terminal ER retention motif, HDEL (H; green). (B) A multiple sequence alignment of FKBP14 orthologs from human (Hsap FKBP14, Accession NP_060416), mouse (Mmus FKBP14, Accession NP_705801), chicken (Ggal FKBP14, Accession XP_418735) and fly (Dmel FKBP14, Accession NP_726074) reveals highly conserved residues (*), conserved substitutions (:) and semiconserved substitutions (.). Underlined regions correspond to the motifs shown in ‘A’.

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Figure 3.2 FKBP sequence alignment. A multiple sequence alignment of small molecular weight FKBPs from human (Hsap FKBP1A [FKBP12], Accession NP_000792; Hsap FKBP2 [FKBP13], Accession AAH03384; Hsap FKBP7 [FKBP23], Accession NP_851939 and Hsap FKBP14 [FKBP22], Accession NP_060416), mouse (Mmus Fkbp1a [FKBP12], Accession NP_032045; Mmus Fkbp2 [FKBP13], Accession NP_032046; Mmus Fkbp7 [FKBP23], Accession NP_034352 and Mmus Fkbp14 [FKBP22], Accession NP_705801), chicken (Ggal FKBP1A, Accession NP_989661; Ggal FKBP7, Accession XP_421981 and Ggal FKBP14 [FKBP22], Accession XP_418735) and fly (Dmel FK506-bp2 [FKBP12], Accession NP_523792; Dmel CG14715, Accession NP_650101 and Dmel FKBP14, Accession NP_726074) reveals highly conserved residues (*), conserved substitutions (:) and semiconserved substitutions (.). The PPIase domain (bold) from Drosophila FKBP14 is highlighted.

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3.4.2 Drosophila FKBP14 is an ER-resident protein

FKBPs have been shown to exhibit distinct patterns of expression. To determine the

subcellular localization of Drosophila FKBP14, I generated antibodies raised against a

Drosophila FKBP14 fusion protein. The specificity of the antibodies was initially confirmed

through an affinity competition assay (data not shown). I then examined the subcellular

localization of FKBP14 in Drosophila Schneider 2 (S2) and Kc167 (Kc) cells, where it is

endogenously expressed (data not shown). I observed Drosophila FKBP14 colocalization with

the ER marker, anti-KDEL, in S2 cells (Fig. 3.3 A - C). In contrast, the Golgi marker, anti-

p120, does not colocalize with FKBP14 in S2 (Fig. 3.3 D - F) or in Kc cells (data not shown).

Because Drosophila FKBP14 colocalizes with an ER marker and contains both a signal

sequence and an ER retention sequence, this demonstrates that FKBP14 resides in the ER.

Figure 3.3 Drosophila FKBP14 localizes to the ER. (A - C) Endogenous FKBP14 (red) is expressed in a punctate pattern in S2 cells, and colocalizes with anti-KDEL (green). Colocalization (yellow) is indicated in the merge. (D - F) FKBP14 (red) does not colocalize with anti-p120 (green) in S2 cells as indicated by the lack of colocalization (yellow) in the merge. DAPI is shown in blue.

3.4.3 FKBP14 mutants exhibit defects in sense organ development

To determine the function of Drosophila FKBP14, I characterized the lethal P-element

insertion referred to as FKBP14EP2019 (Fig. 3.4 A). I also generated deletions in FKBP14

through imprecise excision of the EP(2)2019 insertion. I identified an imprecise excision line,

FKBP14D58, which contains a 2405 bp deletion that partially removes the first exon, leaving the

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first 104 bp intact, and completely removes the second exon, including the translational start site

(Fig. 3.4 A). The FKBP14D58 line fails to complement FKBP14EP2019 (n = 81). As a control, I

generated a precise excision line, FKBP14D34 that is fully viable.

To confirm that FKBP14D58 represents a null mutation in FKBP14, I examined levels of

FKBP14 protein. I found that FKBP14 levels are reduced by over 80% in the original insertion

line, FKBP14EP2019, demonstrating that this represents a hypomorphic mutation in FKBP14 (Fig.

3.4 B). As expected, no protein is detected from the deletion mutant, FKBP14D58, confirming

that this allele represents a null mutation in FKBP14 (Fig. 3.4 B). These data also provide

confirmation that our antibody is specific to Drosophila FKBP14.

Figure 3.4 FKBP14 mutants exhibit reduced FKBP14 levels. (A) An FKBP14 schematic overview (AE013599; Flybase, GBrowse). FKBP14 is flanked by the ORFs of Sara and CG10496. Excision of EP(2)2019 generated FKBP14D58 (imprecise) and FKBP14D34 (precise; not shown) excision alleles. (B) Immunoblot analysis indicates that FKBP14 is absent from FKBP14D58 and reduced from FKBP14EP2019 compared to FKBP14D34 pharate adult extracts. β-tubulin was used as a loading control.

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To further define the effects of a loss of FKBP14 during Drosophila development, I

examined the phenotypes of FKBP14 null mutants. FKBP14 mutants are homozygous lethal. I

found that the predominant mutant phenotype is a loss of larval discs, with 80% of FKBP14D58

mutants lacking imaginal discs (Fig. 3.5 A; n=250), resulting in mid-pupal lethality. FKBP14D58

mutant larval discs and CNS exhibit morphological differences compared to FKBP14D34 third

instar larvae. For instance, FKBP14D58 third instar larval optic lobes appear smaller, and the

ventral nerve cord is longer (Fig. 3.5 C), as compared to wild type (WT; Fig. 3.5 B).

Additionally, FKBP14D58 third instar larval wing and eye discs appear folded and are smaller

(Fig. 3.5 E and G) compared to controls (Fig. 3.5 D and F).

The significant loss of discs that was observed in FKBP14 mutants led us to examine

levels of apoptotic cells in FKBP14 mutant third instar larvae. Using a dye specific to cells that

have undergone apoptosis, known as acridine orange [263], I observed increased levels of

apoptosis in larval ventral nerve cords of FKBP14D58 mutant larvae (Fig. 3.5 I), particularly in

larvae that lack discs (Fig. 3.5 I, inset) as compared to controls (Fig. 3.5 H). Psn null mutants

also exhibit higher levels of apoptotic cells in larval ventral nerve cords, although the pattern of

acridine orange staining differs from FKBP14 mutants (data not shown). Additionally, FKBP14

mutant wing discs exhibit higher levels of apoptotic cells (Fig. 3.5 K, arrow points to the

presumptive wing margin, arrowhead marks the hinge region and bracket highlights a portion of

the presumptive notum region), particularly in small wing discs (Fig. 3.5 K, inset), as compared

to the corresponding regions in controls (Fig. 3.5 J). FKBP14D58 mutant eye discs also display

higher levels of apoptosis (Fig. 3.5 M, arrow marks the morphogenetic furrow), especially in

small discs (Fig. 3.5 M, inset), compared to controls (Fig. 3.5 L). The pattern of acridine orange

staining that was observed in FKBP14D34 wing and eye discs is consistent with what has been

previously observed in wild type tissues [382-384].

A small percentage of FKBP14 mutants die later than the mid-pupal stage of development,

at the pharate or late-pupal stage of development, and this is likely due to perdurance of a

maternal contribution. These mutants display defects in eye development (Fig. 3.6 B, C, E and

F), compared to controls (Fig. 3.6 A and D). FKBP14 developmental expression, including

maternal contribution analysis, is discussed in Chapter 4.

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Figure 3.5 FKBP14 mutants exhibit defects in larval CNS and disc development, and increased levels of apoptosis in third instar larval CNS and discs. (A) Quantitation of third instar larval CNS and discs reveals a significant loss of discs in FKBP14 mutants compared to controls. (B – G) Brightfield images of third instar larval CNS and discs. (B) FKBP14D34 CNS, (D) wing and (F) eye discs appear larger compared to (C) FKBP14 mutant CNS, (E) wing and (G) eye discs. (H – M) Acridine orange (AO) staining of cells undergoing apoptosis in third instar larval CNS and discs. (H) FKBP14D34 third instar larval CNS exhibit normal levels of acridine orange staining in optic lobes and ventral nerve cords. (I) FKBP14D58 third instar CNS exhibit increased levels of acridine orange staining, particularly in ventral nerve cords of third instar larvae lacking discs (inset), compared to controls. (J) Apoptotic cells are detected throughout FKBP14D34 third instar larval wing discs, consistent with WT [384]. Cells that have undergone apoptosis at the presumptive wing margin (arrow), hinge (arrowhead) and presumptive notum (bracket) regions are observed as brightly stained particles. (K) Levels of apoptosis are increased in FKBP14 mutant third instar larval wing discs, particularly in the presumptive wing margin (arrow), hinge (arrowhead) and presumptive notum (bracket) regions, and are significantly increased in small wing discs (inset), as compared to controls. (L) FKBP14D34 third instar larval eye discs exhibit apoptotic cells on either side of the morphogenetic furrow (arrow), consistent with WT [382, 383, 385]. (M) FKBP14 mutant eye discs appear highly folded, and exhibit increased levels of apoptosis in regions surrounding the morphogenetic furrow (arrow), particularly in small eye discs (inset; antennal disc [ad], optic lobe [ol]), as compared to controls. AO staining was performed on unfixed tissues. FKBP14 mutant third instar imaginal discs appear highly folded.

3.4.4 Loss of FKBP14 function impairs Notch signalling at the wing margin and in

proneural clusters

Defects in wing margin specification are observed in FKBP14 mutants (Fig. 3.7 B; arrows

point to mild notching), compared to FKBP14D34 (Fig. 3.7 A). These phenotypes are

reminiscent of those observed in Notch loss-of- function mutants [386]. Notch signalling

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Figure 3.6 FKBP14 mutants display defects in eye development. (A - F) Scanning electron micrographs of adult eye morphology. Bars, 20 uM. (A, D) FKBP14D34 eyes consist of organized ommatidia, separated by interommatidial bristles (arrow in D). (B, C, E and F) Ommatidia are disorganized and bristles are reduced in FKBP14D58 and FKBP14EP2019 pharate adults.

mediates expression of two well-characterized target genes, known as Cut (Ct) and Wingless

(Wg), at the presumptive wing margin [120]. In Notch loss-of- function mutants, expression of

Ct and Wg are reduced [118, 120], whereas enhanced Notch signalling results in expanded Ct

and Wg expression in the wing pouch [387]. I examined whether FKBP14 function is involved

in Notch signalling by analyzing levels of Ct and Wg expression in FKBP14 mutants. I

demonstrate that levels of both Ct and Wg expression are reduced in FKBP14D58 mutants (Fig.

3.7 D and F), compared to controls (Fig. 3.7 C and E). Reduced Ct expression is more extreme

than Wg, similar to what has been shown in Notch loss-of- function alleles [120]. These data are

therefore consistent with a role for FKBP14 in Notch-regulated patterning during wing margin

specification.

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Figure 3.7 FKBP14 is required for Notch target gene expression at the presumptive wing margin. (A) Pharate adult bristle patterning at the wing tip in FKBP14D34 flies. (B) Defects in bristle patterning and mild notching (arrows) are observed in FKBP14D58 wings. (C) FKBP14D34 anterior wing margins exhibit rows of mechanosensory bristles (arrow points to one) and chemosensory bristles (arrowheads), consistent with WT [388]. (D) FKBP14D58 anterior wing margins exhibit reduced numbers of mechanosensory bristles, and socket-to-shaft transformations in some instances (arrow points to a double-shafted bristle, consistent with some Notch loss-of-function phenotypes [388-390]). Additionally, chemosensory bristles are missing. (E) Ct expression is observed in rows of cells at the presumptive wing margin in FKBP14D34 late third instar larval discs (40X), consistent with WT [387]. The region marked by a red asterisk has been magnified (inset; 100X) to show approximately 3 rows of cells that express Ct at the presumptive wing margin. (F) In FKBP14D58 mutants, Ct staining is disrupted across the margin (40X). Cells marked by a red asterisk show a significant reduction in Ct expression (inset; 100X). (G) Wg expression is observed in cells at the presumptive wing margin, and in regions of the wing pouch in FKBP14D34 third instar larval discs, consistent with WT [391-394]. (H) In FKBP14D58

mutant third instar larval wing discs, Wg staining appears mildly reduced in cells at the presumptive wing margin, and is unaltered in cells of the wing pouch. Bars, 20 uM.

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In the presumptive notum region of the wing disc, Notch signalling activates expression of

proteins encoded by the Enhancer of split [E(spl)] complex in cells targeted for an epidermal

cell fate that surround sense organ precursors (SOPs; [112, 395]). To further define the potential

role of FKBP14 in mediating a Notch signal, I analyzed the expression of E(spl) proteins in

FKBP14D58 mutant wing discs. I stained SOPs using anti-Senseless, a nuclear protein that labels

SOPs [396]. I found that the number of SOPs and the levels of E(spl) expression are

significantly reduced in the presumptive wing margin and hinge regions of FKBP14 mutant

wing discs (Fig. 3.8 D - F; magnified in M - O), compared to FKBP14D34 (Fig. 3.8 A - C;

magnified in J - L). In contrast, Psn mutant wing discs display an increase in SOPs and reduced

E(spl) expression (Fig. 3.8 G - I), illustrating a loss of Notch signalling activity at the

presumptive wing margin.

A fate map of a third instar wing disc and adult heminotum illustrates the stereotyped

positions of SOPs (circles) in the presumptive notum region (box) that give rise to adult bristles

(open circles) in WT (Fig. 3.9 A). In FKBP14D34 third instar presumptive nota, I observed 11.3

± 0.21 SOPs that are surrounded by E(spl)-expressing cells (Fig. 3.9 B – E). The dorsocentral

(DC; Fig. 3.9 I), supraalar (SA; Fig. 3.9 J) and postalar (PA; Fig. 3.9 K) regions have been

magnified to illustrate this. A reduction in SOPs is observed in FKBP14D58 presumptive nota

(Fig. 3.9 B, F - H), which is significant (7.4 ± 0.3; n = 20; p<0.0001), and E(spl) expression is

also reduced in cells that surround SOPs, particularly in dorsocentral (DC; Fig. 3.9 L), supraalar

(SA; Fig. 3.9 M) and postalar (PA; Fig. 3.9 N) regions, compared to controls.

A decrease in the number of SOPs has been observed in some Notch gain-of- function

alleles [112, 386, 397]. Since the number of SOPs are reduced in FKBP14D58 third instar larval

wing discs, and expression of proteins of the E(spl) complex is also reduced, indicating a loss of

Notch activity, this suggests that FKBP14 may be involved during SOP specification. I

therefore wanted to determine the effects of a loss of FKBP14 on proneural gene expression in

third instar larval wing discs. Expression of Achaete, a proneural gene that encodes a basic

helix- loop-helix transcriptional activator, in small clusters of proneural cells of the presumptive

notum region confers their potential to adopt an SOP cell fate [111, 398]. A single cell within

these clusters accumulates the highest level of Achaete expression, specifying a neural cell fate,

and signals to surrounding cells via Notch lateral inhibition to adopt an epidermal cell fate [111,

112]. In the presumptive wing margin of third instar larval wing discs, Senseless (Sens) plays a

proneural role in specifying the mechanosensory bristle cell fate, while Achaete (Ac) is required

for survival of the mechanosensory progeny in this region of the wing disc [399]. I found that in

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Figure 3.8 FKBP14 is required for Notch target gene expression in wing pouch and hinge regions. (A - C) Sens (green) expression is detected at the presumptive wing margin and in SOPs of the pouch and hinge (box) regions of FKBP14D34 wing discs. At this exposure, Enhancer of split [E(spl); red] expression is detected in cells surrounding SOPs in the hinge region (magnified in J – L). (D - F) Sens (green) localization is reduced at the presumptive wing margin and in SOPs of the pouch and hinge (box) regions of FKBP14D58 wing discs. Similarly, E(spl) expression (red) is reduced in cells that surround SOPs at the hinge region (magnified in M - O). (G - I) Psn mutants display severe defects in wing disc development, including enhanced Sens (green) expression in an oblong pattern of cells at the presumptive wing margin, and reduced E(spl) expression (red).

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Figure 3.9 FKBP14 is required for SOP development and Notch target gene expression in third instar larval wing discs. (A) Fate map of an imaginal wing disc indicating SOP cells (solid dots) and bristle sites of an adult heminotum (open circles; adapted from [119]). Abbreviations: a, anterior; p, posterior; DC, dorsocentral; NP, notopleural; PA, postalar; SA, supraalar and SC, scutellar. The box highlights the region that is shown in the following panels. (B) Quantitation of SOPs in presumptive nota of third instar wing discs, ± standard error of the mean (represented by error bars). In FKBP14D58 mutants, a decrease in the number of SOPs (7.4 ± 0.3), compared to FKBP14D34 (11.3 ± 0.2), is statistically significant (n = 20; *** p<0.0001). (C – E) E(spl) protein expression (red) is observed surrounding SOPs that have been marked with anti-Sens (green) in FKBP14D34 presumptive nota. The DC (box), SA (arrow) and PA (arrowhead) SOPs have been magnified in (I) DC, (J) SA and (K) PA. (F – H) E(spl) expression (red) is reduced surrounding SOPs (green) in FKBP14D58 mutants. The DC (box; magnified in L), SA (arrow, magnified in M) and PA (arrowhead; magnified in N) regions exhibit significantly reduced E(spl) expression, compared to controls. Bar, 10 uM.

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FKBP14D34 third instar larval wing discs, Ac is expressed in cells that have been marked by

Sens in the wing pouch (Fig. 3.10 A – C) and in cells of the presumptive notum (Fig. 3.10 G – I;

arrowhead points to a single proneural cluster). In the presumptive wing margin of FKBP14D58

mutants, levels of Ac and Sens expression are reduced (Fig. 3.10 D – F). Additionally,

FKBP14D58 mutants exhibit a mild reduction in Ac expression in cells of the presumptive notum

(Fig. 3.10 J – L, arrowhead marks a single proneural cluster), as compared to controls. Since

Psnw6rp exhibited reduced Notch signalling activity, consistent with previous reports [56, 84], I

examined levels of Sens and Ac expression in third instar larval wing discs. I observed

increased levels of Sens and Ac in the presumptive notum region of Psnw6rp wing discs (Fig.

3.10 M – O), compared to FKBP14D34 and, surprisingly, compared to FKBP14D58 mutant wing

discs. These data suggest that FKBP14 may affect SOP development upstream of the Notch

signalling pathway.

A loss of proneural gene activity has been shown to reduce SOP numbers in presumptive

notum regions of third instar larval wing discs, resulting in balding phenotypes of adult nota

[315]. Since I observed a mild reduction in Ac expression and decreased SOP numbers in

FKBP14D58 mutant wing discs, I examined whether FKBP14D58 mutants displayed defects in

bristle patterning of adult nota. In FKBP14D34 nota, microchaetae (arrow) and macrochaetae

(arrowhead) are evenly dispersed (Fig. 3.11 A and D). In FKBP14 mutants, I observe an almost

complete reduction of microchaetae (arrows) and a significant reduction in macrochaetae

(arrowheads) in pharate adult nota (Fig. 3.11 B, C, E and F), compared to FKBP14D34. This

suggests that FKBP14 may be required for proneural gene activity in presumptive nota during

SOP determination (discussed in Chapter 6). Interestingly, overexpression of a Notch negative

regulator known as numb [400], or loss of a Notch regulator known as neuralized [388], also

resulted in extreme notum balding [388, 389], similar to what was observed in FKBP14 mutants.

This indicates that a loss of FKBP14 function may result in reduced Notch activity.

3.4.5 FKBP14 is not required for Notch trafficking to the plasma membrane

Since FKBP14D58 mutants exhibit significant defects in Notch signalling, I sought to

determine how FKBP14 affects the Notch pathway. In vertebrates, Notch is synthesized in the

ER and then processed in the Golgi, leading to the formation of a heterodimeric receptor at the

plasma membrane [401], whereas in Drosophila, the majority of Notch protein at the plasma

membrane is uncleaved [98]. Notch activation occurs at the cell surface upon interaction with

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Figure 3.10 FKBP14 function is involved in proneural gene expression in third instar larval wing discs. (A – C) Sens (green) and Ac (red) expression is observed in cells at the presumptive wing margin and hinge regions of FKBP14D34 wing discs. Anterior is to the left, ventral is up. Ac localizes to the presumptive anterior wing margin. (D – F) Sens and Ac expression levels are reduced at the presumptive wing margin of FKBP14D58 mutants, compared to controls. (G – I) In FKBP14D34 presumptive nota, Ac (red) localizes to cells that comprise proneural clusters (arrowhead marks a single cluster). SOPs have been labelled with Sens (green). (J – L) In FKBP14D58 mutants, Ac (red) expression is mildly reduced in some proneural clusters (arrowhead marks one), compared to controls. SOPs have been marked with Sens (green). (M – O) Psn mutants display severe defects in Sens (green) and Ac (red) expression in cells of the presumptive nota.

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Figure 3.11 FKBP14 is required for bristle formation in adult nota. (A and D) FKBP14D34 nota display organized microchaetae (arrows) and macrochaetae (arrowheads). (B, C, E and F) FKBP14 mutant pharate adults exhibit a severe reduction in the number of bristles on the thorax, compared to controls. In FKBP14D58, two microchaetae are shown (arrows), and in FKBP14EP2019, one microchaete (arrow) and one putative macrochaete (arrowhead) are shown.

its ligands. Subsequent g-secretase proteolysis of the protein, which may occur in the endocytic

pathway [402], generates a Notch intracellular domain that translocates to the nucleus [88] and

initiates transcription of downstream targets [120]. Notch can therefore be regulated during its

synthesis, transport to the membrane and proteolysis. FKBP14 is an ER-resident protein that is

thought to play a role in protein folding, therefore I wanted to examine whether Notch

trafficking to the membrane was affected in FKBP14 mutants. I observe that Notch is

predominantly localized at the plasma membrane in FKBP14D58 wing discs (Fig. 3.12 B),

compared to controls (Fig. 3.12 A). Moreover, the distribution of Notch in FKBP14 mutants is

similar to that observed in Psn null mutant wing discs, where Notch cleavage is affected after it

has reached the plasma membrane (Fig. 3.12 C). Notch plasma membrane expression is also

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intact in cells (arrowheads) that border the presumptive wing margin (arrows) in FKBP14D34 and

FKBP14 mutant wing discs (Fig. 3.12 D and E). Notch trafficking to the plasma membrane is

therefore not defective in FKBP14 mutants. Taken together with previous observations that

levels of Notch target genes are reduced in FKBP14 mutants, these data indicate that FKBP14

may affect Notch signalling downstream of receptor trafficking to the cell surface.

Figure 3.12 FKBP14 functions downstream of Notch trafficking to the plasma membrane. (A - C) Notch is detected at the plasma membrane in FKBP14D34, FKBP14D58 and Psnw6rp presumptive nota regions, using an antibody that recognizes the Notch extracellular domain (C458.2H; DSHB). (D and E) Notch extracellular expression is observed at the plasma membrane in cells (arrowheads) of the presumptive wing margin (arrows) in (D) FKBP14D34 and (E) FKBP14D58 wing discs. Bars, 10 uM.

3.4.6 FKBP14 mutants genetically interact with Notch and Psn

If FKBP14 is mediating Notch activity, then a mutation in FKBP14 may modulate the

phenotypes caused by a Notch loss-of- function allele. To examine this interplay, I analyzed

whether FKBP14 mutants could genetically interact with a Notch deficiency that causes

notching of the distal wing margin due to haploinsufficiency (Fig. 3.13 B – D).

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Transheterozygotes of the Notch deficiency, Df(1)N-8, and either FKBP14D58 or FKBP14EP2019

exhibit enhanced wing notching (Fig. 3.13 E and F), compared to controls (Fig. 3.13 A – D),

thereby confirming a link between FKBP14 and Notch (n 10 for each genotype). Enhanced

wing notching is also observed in Df(1)N-8; Psnw6rp transheterozygotes (Fig. 3.13 G), similar to

our previous analysis in which we determined that Psn mutations reduced Notch activity [56].

These results indicate that a loss of FKBP14 function reduces Notch signalling activity and

suggest that FKBP14 and Psn may function together to propagate a Notch signal.

Figure 3.13 Quantitation of the genetic interactions between FKBP14 and Notch. (A) WT flies have a smooth wing margin (arrowhead). (B - G) Quantitation of the wing blade defects observed in each cross (representative images are shown at the top of the table) are displayed in the panels to the right of each genotype. (B) Distal wing blade notching observed in Df(1)N-8/FM7 (DfN-8/FM7) is reduced when outcrossed to the WT strains, OreR and FKBP14D34 (D34): (C) DfN-8/+; OreR and (D) DfN-8/+; D34. Wing notching is enhanced in (E) DfN-8; FKBP14D58, (F) DfN-8; FKBP14EP2019 and (G) DfN-8; Psnw6rp transheterozygotes, compared to the controls in C and D (n 10).

Psn holoprotein is synthesized in the ER and rapidly undergoes endoproteolysis to

generate N- and C-terminal fragments (NTF, CTF) that are required for g-secretase function

[32]. Psn is thought to comprise the catalytic activity of g-secretase [35] that is involved in

Notch cleavage. To determine if FKBP14 regulates Psn function, I tested the ability of FKBP14

mutants to genetically interact with Psn using the GAL4/UAS system [258]. I show that

FKBP14D58 suppressed Psn-dependent bristle phenotypes (Fig. 3.14 A – E) and FKBP14EP2019

suppressed notched wing phenotypes (Fig. 3.14 F – H) that result from Psn overexpression

during larval development. Psn overexpression phenotypes are similar to those observed in

Notch loss-of-function mutants and may result from dominant-negative effects [263]. Together,

these results are consistent with FKBP14 functioning within the Notch pathway.

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3.4.7 FKBP14 colocalizes and binds with Psn in the ER

A genetic interaction between FKBP14 and Psn may represent an interaction between the

two gene products. I examined whether FKBP14 and Psn colocalize in Kc cells, where both

proteins are endogenously expressed (data not shown). Drosophila Psn localizes to the

cytoplasm of neurons [56] and Kc cells, and colocalizes with FKBP14 in the ER (Fig. 3.14 I –

K), indicating that the two proteins may associate within the early secretory pathway. I then

performed co- immunoprecipitation experiments to determine whether FKBP14 and Psn are

found within a protein complex. When Myc-tagged Psn (Psn-Myc) fusion proteins are

overexpressed in S2 cells, I detect Psn holoprotein without increasing levels of PsnNTF,

consistent with other reports [34, 403]. In this context, I find that endogenous FKBP14 co-

immunoprecipitates with full- length Psn-Myc (Fig. 3.14 L), demonstrating that these proteins

Figure 3.14 FKBP14 genetically and physically interacts with Psn. (A) A schematic showing the bristle positions (circles) of a WT nota, with the highlighted region (box) shown in the panels to the right. (B) WT flies have four scutellar bristles (arrow points to one). Bar, 100 uM. (C) The ectopic bristle phenotype (arrowhead points to one) observed in a pannier-GAL4/UAS-Psn transgenic line is (D) unaltered in FKBP14D34 and (E) suppressed in FKBP14D58 heterozygotes. (F) WT flies have a smooth distal wing margin (arrowhead). (G) Wing notching observed at the distal wing margin in a cut-GAL4/UAS-Psn recombinant line is (H) suppressed by FKBP14EP2019 heterozygotes. (I - K) FKBP14 (red) and Psn (green) partially colocalize (yellow; merge) in Kc cells. DAPI is shown in blue. (L) Endogenous FKBP14 co-immunoprecipitates with Psn-Myc holoprotein and not with vector alone (top blot). Psn-Myc is detected as a holoprotein (PsnFL-Myc), and endogenous Psn N-terminal fragments (PsnNTF) are also observed (middle blot). β-tubulin was used as a loading control, and detects non-specific (ns) bands (bottom blot, left). Anti-Myc detects IgG bands (bottom blot, right).

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are found within a complex. I did not detect FKBP14 using empty vector as a control (Fig. 3.14

L), indicating that the interaction between FKBP14 and Psn holoprotein is specific.

Furthermore, I was unable to detect an interaction between endogenous FKBP14 and

endogenous PsnNTF (Fig. 3.14 L) or PsnCTF (data not shown), suggesting that FKBP14 may not

be required to stabilize the active heterodimer. Our data demonstrate that FKBP14 binds Psn

within the ER, and suggest that FKBP14 may play a role in regulating Psn.

3.4.8 FKBP14 function maintains Psn protein levels

To determine how FKBP14 regulates Psn, I examined Psn protein levels in FKBP14

mutants using an anti-Psn antibody. I found that levels of endogenous Psn are reduced up to

90% in FKBP14 mutants compared to controls (Fig. 3.15 A). The effects on Psn levels are post-

transcriptional as levels of Psn RNA are not reduced in FKBP14 mutants (Fig. 3.15 B). I also

examined whether FKBP14 levels were affected by a loss of Psn protein and found no

significant differences (Fig. 3.15 A), suggesting that FKBP14 acts upstream of Psn.

Figure 3.15 Loss of FKBP14 affects Psn protein levels. (A) Psn protein expression is absent in Psn mutant extracts, and is reduced up to 90% in FKBP14 mutant extracts, which are protein-null. FKBP14 expression is unaffected in Psn mutant extracts. β-tubulin shows equal loading. (B) RT-PCR analysis of Psn transcripts shows reduced Psn levels in Psn mutant extracts compared to FKBP14 mutants and controls. Gapdh amplification was performed as a loading control.

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3.5 Discussion

In this Chapter, I have characterized the function of FKBP14 in Drosophila. Sequence

analysis shows that Drosophila FKBP14 exhibits a single PPIase domain, a calcium-binding EF

hand and a C-terminal HDEL endoplasmic reticulum retention motif, similar to its mammalian

ortholog. I demonstrate that FKBP14 loss-of- function mutants are lethal and give rise to severe

defects in imaginal disc development that result in eye, wing and notum phenotypes. Defects in

wing and notum development may be caused by reduced proneural gene expression and reduced

Notch target gene expression, downstream of Notch targeting to the plasma membrane. I also

show that FKBP14 genetically interacts with both Notch and Psn, a critical component of γ-

secretase, and that FKBP14 and Psn colocalize in the ER, where they form a protein complex.

Finally, I determined that Psn protein levels are significantly reduced in FKBP14 mutants,

consistent with a role for FKBP14 in regulating Psn protein folding or stability.

Presenilins function as part of the multimolecular γ-secretase complex that cleaves single-

pass transmembrane proteins such as Notch and APP [5, 99]. At present the core g-secretase

complex is composed of at least four proteins: Presenilin (Psn), nicastrin (Nct), anterior pharynx

defective 1 (Aph-1) and presenilin enhancer 2 (Pen-2) [28, 31]. Presenilin is synthesized as an

~50 kDa precursor protein within the ER that rapidly undergoes endoproteolytic cleavage within

a cytoplasmic loop to generate amino and carboxy terminal fragments that comprise the catalytic

activity of γ-secretase [32, 35]. The holoprotein, unlike the N- and C-terminal fragments, is

highly unstable and must be incorporated into a larger complex for stabilization [33].

Unassembled g-secretase components have been shown to interact with the factor Retention in

endoplasmic reticulum 1 (Rer1), that binds immature nicastrin [42] and unassembled presenilin

enhancer 2 for retrieval to the ER [70]. FKBPs were originally believed to play a general role in

protein folding and assembly throughout the secretory pathway, although recent studies have

demonstrated distinct FKBP14 subcellular localization and interaction with specific protein

targets in yeast [209]. In Drosophila, I demonstrate that an interaction between FKBP14 and

Psn is essential for development.

I generated an FKBP14 null mutant that is lethal during the mid-pupal stage of

development, although some mutants develop until the late-stage of pupal development, likely

due to perdurance of a maternal contribution. In FKBP14D58 homozygotes, the predominant

phenotype is a loss of larval discs, with 80% of homozygotes lacking imaginal discs (n = 250),

resulting in mid-pupal lethality. I found that levels of apoptosis are increased in FKBP14D58

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larval CNS and eye and wing discs, compared to controls. These data suggest that FKBP14

function may be involved in apoptosis regulation during larval development.

A small percentage of FKBP14 mutants die late during pupal development, and display

defects in bristle sense organ development in adult eyes, nota and wings. The wing defects

observed in FKBP14 mutant pharate adults included loss of bristles, bristle twinning and

notching at the distal wing margin. These defects phenocopy what has been observed in some

Notch loss-of-function alleles. For instance, overexpression of a negative regulator of Notch

known as numb [400] caused a loss of mechanosensory bristles and bristle twinning at the adult

wing margin [389], and loss of an E3 ubiquitin ligase that regulates the Notch pathway, known

as neuralized [404], caused bristle twinning defects in adult wings [388]. Patterning of the wing

begins during the larval stage and requires coordination of various signal transduction pathways.

For instance, Cut (Ct) and Wingless (Wg) respond to cues from Notch, Delta and Serrate to

define the presumptive wing margin [118, 120]. Notch signalling specifically maintains Ct

expression at the margin whereas Ct may be required to maintain Wg transcription [120]. In

Notch loss-of-function mutants, reduced Ct and Wg expression is observed [118, 120], whereas

enhanced Notch signalling results in expanded Ct and Wg expression in the wing pouch [387]. I

determined that levels of both Ct and Wg expression are reduced in FKBP14D58 mutants, and

that reduced Ct expression is more extreme than Wg, similar to what has been shown in Notch

loss-of-function alleles [120]. Together, these data suggest that FKBP14 may be required for

Notch signalling in developing wing discs.

FKBP14 mutants also exhibit defects in sense organ precursor (SOP) development. In the

presumptive notum region, SOP determination requires the interplay between the achaete-scute

proneural gene complex [398] and Notch signalling [112]. Expression of Achaete, encoding a

basic helix- loop-helix transcription factor, in small clusters of proneural cells allows them to

adopt an SOP cell fate [112]. A single SOP cell within each proneural cluster accumulates

achaete-scute expression and signals to surrounding cells via Notch-mediated lateral inhibition

to adopt an epidermal cell fate [96, 111]. Reduced Achaete expression associates with a

reduction in SOP levels [112, 315, 395], and achaete-scute mutants exhibit a balding phenotype

in adult nota [315]. Similarly, loss of adult nota bristles has been observed in some Notch loss-

of- function alleles [388, 389]. In the presumptive wing margin, the zinc-finger transcription

factor Senseless acts as a proneural gene to confer neuronal identity to cells of the

mechanosensory cell lineage, while achaete-scute proneural gene activity is required to specify

the chemosensory cell fate [399]. FKBP14 mutants exhibited a significant loss of SOPs in third

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instar larval wing discs, which associated with a significant loss of adult nota and wing bristles,

suggesting that FKBP14 may be involved in proneural activity. Consistent with this model, I

found that Achaete and Senseless expression levels were reduced in proneural clusters of the

presumptive notum region, and in cells at the presumptive wing margin of FKBP14 mutant wing

discs, compared to controls. To determine the effects of a loss of FKBP14 function on Notch

signalling in third instar larval wing discs, I examined the expression of proteins encoded by the

Enhancer of split [E(spl)] complex in cells targeted for an epidermal cell fate that surround

SOPs [112, 395]. I observed significantly reduced E(spl) expression in FKBP14 mutant

presumptive nota and additionally, in cells of the hinge region, indicating a loss of Notch

activity. Together, these data suggest that the loss of pharate adult bristles in FKBP14 mutants

may be due to reduced proneural gene expression and a defect in Notch signalling activity.

In vertebrates, Notch is first synthesized in the endoplasmic reticulum and then processed

by furin proteases in the Golgi apparatus, leading to the formation of a heterodimeric receptor at

the plasma membrane [401], whereas in Drosophila, the majority of Notch protein at the plasma

membrane is uncleaved [98]. Notch activation occurs at the cell surface upon interaction with

its ligands, and subsequent g-secretase proteolysis of the protein within the endocytic pathway

[402]. This generates a Notch intracellular domain that translocates to the nucleus [88] and

initiates transcription of downstream targets [120]. Notch can therefore be regulated during its

synthesis, transport to the membrane and proteolysis. I show that Notch was predominantly

localized at the plasma membrane in FKBP14D58 wing discs, and the distribution of Notch was

similar to that observed in Psn mutant (Psnw6rp) wing discs, where Notch cleavage is affected

after it has reached the plasma membrane [402]. Notch plasma membrane expression is also

intact in cells that border the presumptive wing margin in FKBP14 mutant wing discs. Since I

show that levels of Notch target genes are reduced in FKBP14 mutants, this indicates that

FKBP14 may affect Notch signalling downstream of receptor trafficking to the cell surface,

such as Psn-dependent γ-secretase processing of Notch. Previous studies have shown that Psn

dominant-negative mutants give rise to fused ommatidia, loss of eye bristles and wing notching

[263], similar to what I observed in FKBP14 mutants. Additionally, Psn downregulation via

DAPT treatment, which inhibits γ-secretase activity, reduces Ct and Wg expression at the

presumptive wing margin [392] in a manner analogous to what I described here. I therefore

examined whether FKBP14 could genetically interact with Notch and Psn. My analysis showed

that FKBP14 null mutants enhanced Notch loss-of-function defects, and suppressed Psn

overexpression phenotypes. These results are consistent with FKBP14 functioning within the

Notch signalling pathway, and suggest that FKBP14 may be required to regulate Psn activity.

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The genetic interaction between FKBP14 and Psn may represent an interaction between

both proteins. I demonstrate that in Kc cells, where both proteins are endogenously expressed,

Psn partially co-localized with FKBP14 in the ER. At this stage of the secretory pathway, the

predominant form of Psn is believed to be holoprotein [42, 405], and thus FKBP14 may promote

its proper folding prior to endoproteolysis or until it can assemble with other components of the

g-secretase complex. I found that endogenous FKBP14 co- immunoprecipitated with full- length

Psn-Myc, confirming that both proteins are found within a complex. Furthermore, I was unable

to detect an endogenous interaction between FKBP14 and PsnNTF or PsnCTF (data not shown),

indicating that FKBP14 may not be required to stabilize the active heterodimer. In agreement

with this, Psn endoproteolysis is believed to occur at later stages of the secretory pathway,

possibly away from FKBP14 [80, 405]. Taken together, these data demonstrate that FKBP14

binds Psn within the ER, possibly to regulate Psn.

To determine whether FKBP14 is required for Psn function, I examined the effects of a

loss of FKBP14 on Psn protein stability. I determined that Psn protein levels are reduced up to

90% in FKBP14 mutants compared to controls. These effects are post-transcription, since Psn

RNA levels are similar in both FKBP14D34 and FKBP14D58 extracts. Additionally, FKBP14

protein levels were not affected by a loss of Psn function, suggesting that FKBP14 acts upstream

of Psn. These data also suggest that the defects in Notch signalling that were observed in

FKBP14 mutants may be due to reduced Psn protein levels and thus, g-secretase activity.

Altogether, my analyses demonstrate that FKBP14 function is essential during Drosophila

imaginal disc development. FKBP14 function may be pleiotropic, as illustrated by the

numerous developmental processes that are affected by a loss of FKBP14 activity. Since Psn-

dependent g-secretase activity is required to cleave numerous single-pass transmembrane

proteins, involved in a variety of signalling pathways, it will be interesting to determine if

FKBP14 also plays a role in additional developmental processes that are regulated by Psn.


Enhancer of split and Senseless antibodies were provided by Dr. S. Bray and Dr. H. Bellen

respectively. The co- immunoprecipitation assay was modified from a previous protocol as

described [376].

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CHAPTER 4

ANALYSIS OF FKBP14 FUNCTION DURING DEVELOPMENT

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4.1 Summary

FK506-binding proteins (FKBPs) have been implicated in protein folding, assembly and

trafficking. FKBP14 orthologs from Neurospora crassa function as chaperones whereas

bacterial FKBP14 orthologs are involved in cold-adaptation. Mammalian FKBP14 has yet to be

characterized. Here, I demonstrate that expression of Drosophila FKBP14 is dynamic

throughout development, and is specifically localized to nurse cells and oocytes in developing

egg chambers, embryonic hemocytes and larval CNS. I further demonstrate that enhanced

FKBP14 expression results in increased Psn protein levels in vivo, and a mild phenocopy of Psn-

dependent Notch-related defects in bristle development. Since Psn function is integral for g-

secretase processing of Notch, I examined the effects of co-overexpression of both proteins. I

demonstrate that ectopic expression of FKBP14 and Psn transgenes results in synergistic bristle

phenotypes, suggesting that both proteins function within the same pathway.

4.2 Introduction

The FK506-binding protein (FKBP) family is conserved throughout Metazoa. Numerous

studies show that FKBPs encode highly conserved peptidyl-prolyl cis-trans isomerase (PPIase)

domains that are required for protein folding activity [181, 188]. In vertebrates, FKBP12 binds

to and modulates the gating properties of two calcium release channels, known as IP3R and

Ryanodine receptor [195, 372]. In yeast, FKBP13 orthologs are upregulated in the ER in

response to unfolded proteins [205, 208], whereas FKBP14 orthologs have been shown to bind

via the PPIase domain to chaperone complexes in the ER that contain the molecular chaperones

BiP and PDI [201, 209]. This in turn enhances the chaperone activity of yeast FKBP14

orthologs [201, 209]. Mammalian FKBP38 targets Bcl-2 and Bcl-XL to mitochondria thereby

inhibiting apoptosis [179]. Recent studies also show that FKBP38 forms a complex with

Presenilins, although FKBP38 lacks PPIase activity [154, 179]. Larger FKBPs include the

cytosolic FKBP52, which mediates androgen receptor transcriptional activity in vivo [200].

Thus, FKBPs can exhibit distinct subcellular localization and protein-protein interactions.

In Drosophila, I demonstrate that FKBP14 is broadly expressed throughout development,

and localizes to specific cells in ovaries, embryos and larval ventral nerve cords. My analysis

shows that FKBP14 ectopic expression results in increased Psn protein levels in vivo. Since I

previously determined that FKBP14 forms a complex with Psn (described in Chapter 3), I

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analyzed co-overexpression of both proteins, and demonstrate that this results in synergistic

bristle phenotypes in adult nota. Altogether, my data demonstrate FKBP14 expression during

development, and supports a model whereby FKBP14 functions within the same pathways that

require Psn activity.


4.3.1 Plasmids

The cDNA encoding the FKBP14-RA isoform, amplified from the EST clone GH08925,

and the cDNA encoding the Psn-RA isoform, amplified from the EST clone LD23505, were

engineered to express C-terminal Myc or V5 epitopes and 5’ Kozak sequences. EST clones

were obtained from the Berkeley Drosophila Genome Project. The resulting fragments were

cloned into the EcoRI and NotI sites of the pAc5.1/V5-His plasmid (Invitrogen) to generate

pFKBP14-Myc, pFKBP14-V5, pPsn-Myc and pPsn-V5 plasmids for constitutive expression in

S2 cells, or the KpnI site of pUAST to generate UAS-FKBP14-Myc (FM2 or FM3), UAS-Psn-

Myc (PM5 or PM9) and V5-tagged (not discussed here) constructs. Transgenic lines carrying

UAS-FKBP14 and UAS-Psn constructs were generated by J. Hughes, using standard P-element

transformation procedures [261]. Independent insertions were balanced over SM5 or TM3.


The pannier-GAL4 (P[GawB]pnrMD237/TM3, Ser; 3039) and tubulin-GAL4 (P[tubP-

GAL4]/TM3, Sb; 5138) lines were obtained from the Bloomington Stock Center and scabrous-

GAL4 (sca537.4) has been described in FlyBase [57]. These lines were used to overexpress UAS

transgenes at 29°C. UAS-PM5, UAS-PM9, UAS-FM2 and UAS-FM3 were generated in this

study and UAS-EGFP (P[UAS-GFP.S65T]T2; 1521) was obtained from the Bloomington Stock

Center. snail-twist/CyO (3299), the homozygous viable viking-GFP (G454) and w1118 (3605)

lines were obtained from the Bloomington Stock Center. FKBP14 imprecise excision alleles

(Chapter 3) were recombined to pnrMD237 and UAS-PM5 alleles to generate FKBP14D58; pnr-

GAL4 and FKBP14D58; UAS-PM5 lines. These lines were crossed and allowed to lay eggs for

two days. Emerging larvae were then transferred to 29°C for Psn-Myc overexpression in an

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FKBP14D58 homozygous genetic background. As a control, the precise excision allele

FKBP14D34 (Chapter 3) was recombined to pnrMD237 and UAS-PM5 alleles, and genetic crosses

were conducted similarly. Embryo collections from the wild type strain, Oregon-R maintained

at 25°C, were used for FKBP14 expression analysis in embryos. FKBP14D34 was used to

examine endogenous FKBP14 localization in ovaries, third instar larvae and larval CNS.


Extracts for Western analysis were prepared using standard procedures as described (van

de Hoef et al. submitted). S2 cells harvested 48 h post-transfection, embryos, larvae, pupae,

pharates and adult tissues were homogenized in RIPA buffer (1% NP40, 0.5% sodium

deoxycholate, 0.1% SDS, 0.02 molar Tris-Cl pH 8.0, 0.137 molar NaCl, 10% glycerol, 2 uM

EDTA) supplemented with protease inhibitors (Roche). Lysates were centrifuged at 10,000g for

15 minutes and supernatants were analyzed. SDS-polyacrylamide gel electrophoresis (10%

SDS-PAGE) and immunoblot analyses were carried out as described [262]. The primary

antibodies used were rabbit anti-Psn-NTF [262] and rat anti-FKBP14 (van de Hoef et al.

submitted). Experimental input was determined using mouse anti-b-tubulin and mouse anti-

JLA20 (Actin) both from the Developmental Studies Hybridoma Bank. HRP secondary

antibodies were used (Jackson ImmunoResearch; 1:10 000). All blots were performed in

triplicate. Quantitation was performed using a Fluorchem 8000 Gel Documentation System and

Alpha Innotech software (San Leandro, CA).

4.3.4 Immunostaining

Immunostaining was performed using standard procedures [377-380]. Embryos were

collected at the indicated time intervals. Larval imaginal discs and CNS were fixed with 3%

paraformaldehyde in PBS at RT for 30 min, washed in 0.1% Triton and PBS (PBT) and blocked

in 5% normal donkey serum (NDS) diluted in PBT for 1 hour. Tissues were then incubated with

primary antibodies diluted in PBT containing 5% NDS for 16 hours at 4°C, washed in PBT,

followed by incubation with secondary antibodies in PBT for 1 hour at RT. After 3 washes in

PBT, samples were mounted in Dako (DakoCytomation). S2 cells were fixed with 4%

paraformaldehyde in PBS at RT for 10 min, washed in PBS and non-specific interactions were

blocked in 1% normal donkey serum (NDS) and 3% bovine serum albumin diluted in PBT for 1

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hour. Cells were incubated with primary antibodies diluted in blocking solution for 1 hour at

RT, followed by washes in PBT and then incubation with secondary antibodies in PBT for 1

hour at RT. After 3 washes in PBT, samples were mounted in Dako (DakoCytomation).

Ovaries were dissected in cold PBS, pH 7.0, fixed in 5% paraformaldehyde in PBS for 15

min at room temperature (RT), washed twice in PBS, followed with two washes in PBT (PBS

with 0.3% Triton X-100). To reduce non-specific interactions, ovaries were blocked in 5%

normal donkey serum and 0.2% bovine serum albumin in PBT 2x 30 min at RT. Incubation of

primary antibodies, diluted in block, was performed overnight at 4ºC with gentle nutation. The

ovaries were rinsed in PBT, blocked for 1 h at RT, and incubation of secondary antibodies,

diluted in block, followed overnight at 4ºC. The ovaries were rinsed in PBS 3x 10 min, treated

with 400 mg/ml RNase A (Sigma) for 2 h at RT, rinsed 2x 10 min, and stained with the nuclear

dye ToPro-3 (ToPro; Invitrogen; 1:200) in PBS for 15 min. The samples were washed in PBS,

and mounted in DABCO mounting media.

The following primary antibodies were used: rat a-FKBP14 (1:500), rabbit a-Srp [[406];

1:500], rabbit a-GFP (Invitrogen; 1:1000), rabbit a-PsnCTF (as described in Chapter 3; 1:200).

A488 and Cy3 secondary antibodies (Jackson ImmunoResearch; 1:1000) were used. DAPI was

used at 1:5000. Images were obtained using a Zeiss LSM510 META Confocal Microscope or a

Leica DMRA2 Fluorescence Microscope and processed in Photoshop CS and Illustrator CS.

4.3.5 Cell culture

S2 cells were maintained at RT in Schneider’s media supplemented with 10% fetal bovine

serum. Cells were transfected with 3 ug of plasmid DNA using Cellfectin (Invitrogen) at RT.

4.4 Results

4.4.1 FKBP14 is detected in all stages of development

To determine if FKBP14 was required for early patterning, I examined maternal

contribution of FKBP14 in unfertilized eggs and its expression in early embryos. FKBP14

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maternal protein expression is observed in extracts from FKBP14D34 unfertilized eggs and

embryos 0-3 hours (0-3 h) after egg laying (Fig. 4.1; left blot). I further demonstrate that

FKBP14 levels are constant in FKBP14D34 extracts of 0-3 h and 3-6 h embryos, increase in 6-9 h

and 16-18 h embryonic extracts, then stabilize in larval and pupal extracts (Fig. 4.1; middle

blot). A decrease in FKBP14 levels observed in FKBP14D34 adult extracts may be due to

unequal loading. In FKBP14D58 mutants, levels of FKBP14 proteins are reduced in larval and

pupal extracts (Fig. 4.1, right blot), compared to controls. Since FKBP14 maternal expression is

detected in unfertilized egg extracts, detection of FKBP14 in FKBP14 null mutant embryo

extracts (Fig. 4.1; right blot) is due to maternal contribution. This blot was overexposed

compared to the control blot in order to illustrate the faint FKBP14 expression in pupal extracts.

Figure 4.1 FKBP14 protein expression is required during development. Western blot analysis of unfertilized egg extracts shows maternal expression of FKBP14 protein (left blot). In FKBP14D34 staged embryos, larvae, pupae and adults, FKBP14 expression is detected in 0-3 h and 3-6 h extracts, and is relatively constant from mid-stage embryogenesis (6-9 h) until the pharate stage of development (middle blot). A decrease in FKBP14 levels in adult flies may be due to unequal loading. Perdurance of maternal FKBP14 protein is detected in FKBP14D58 embryo extracts, whereas FKBP14 levels are significantly reduced in larval and pupal extracts (right blot) compared to controls. The exposure time was doubled for this blot to illustrate perdurance of maternal FKBP14 proteins in FKBP14D58 pupal extracts. A putative increase in FKBP14 protein levels in FKBP14D58 L3 extracts could be due to unequal loading. Similarly, the high lipid concentration of larval tissue extracts may account for an observed decrease in β-tubulin levels in FKBP14D34 L1 and L2 and FKBP14D58 L2 extracts, possibly due to aggregation of proteins.

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4.4.2 FKBP14 is broadly expressed in developing egg chambers and early-stage embryos,

and is specifically localized in late-stage embryonic hemocytes

To assess the role of FKBP14 during oogenesis, I examined FKBP14 expression in

developing egg chambers. I observed FKBP14 localization in the germarium, and in early-stage

egg chambers (data not shown). I also demonstrate that FKBP14 localizes to nurse cells,

oocytes and follicle cells in mid-stage egg chambers (Fig. 4.2 A - C). Additionally, FKBP14 is

expressed in follicle cells that surround the oocyte in late-stage egg chambers (data not shown).

This pattern of expression is similar to what has been previously observed with FKBP14-GFP

fusion proteins expressed from the endogenous loci [407].

Figure 4.2 FKBP14 expression is detected in follicle cells, nurse cells and oocytes in mid-stage egg chambers. (A) Endogenous FKBP14 protein localizes to follicle cells, nurse cells and oocytes in stage 6 – 8 egg chambers. (B) ToPro staining of nuclei is shown in blue, and nurse cell nuclei staining is most prominent. (C) Overlay. Bar, 10 uM.

I then examined FKBP14 expression in WT embryos at all stages of development, to

assess the requirement of FKBP14 throughout embryogenesis. I found that in syncytial

blastoderms, FKBP14 expression is detected throughout the cytoplasm and surrounds

chromosomes that have been stained with DAPI (Fig. 4.3 A - C). In mid-stage embryos, 6 h

after egg laying or at stage 11 according to the Bownes stages of embryo development [408,

409], a structure termed the stomodeum (arrow) is visible from the ventral anterior pole of the

embryo towards the gut (Fig. 4.3 D). Additionally, specialized immune cells termed pro-

hemocytes begin to proliferate from the head mesoderm [139]. These cells express a GATA

transcription factor known as Serpent, that has also been shown to localize to the midgut [406].

I demonstrate that FKBP14 and Serpent are expressed in pro-hemocytes, although there is a

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subset of cells in the dorsal region of the embryo that specifically express Serpent (Fig. 4.3 D –

F). FKBP14 staining in CNS tissue may be specific, or may be due to background staining.

At 16 – 18 h of development, or Bownes stages 16 and 17, germband retraction has ended

and organogenesis is complete [408, 409]. Intersegmental furrows (arrowheads point to two)

have also developed (Fig. 4.3 G). At this stage, Serpent expression is detected in differentiated

pro-hemocytes, known as plasmatocytes and crystal cells, in addition to fat body cells that are

Figure 4.3 FKBP14 localization in embryonic hemocytes and fat body. (A - C) In WT (OreR) syncytial blastoderm embryos, FKBP14 expression (red) surrounds nuclei that are stained with DAPI, shown in blue. 1-2 hours (1-2 h) indicates time after egg laying. (C) Overlay is shown. (D - F) In a lateral view of 5-7 h WT embryos, marked by the appearance of the stomodeum (arrow), FKBP14 staining (red) is detected in cells migrating from the head mesoderm that express Serpent (Srp; green). (F) Overlay is shown. (G - I) In a lateral view of 16-18 h WT embryos, FKBP14 staining (red) localizes to hemocytes that migrate throughout the hemolymph that express Srp (green). A subset of cells express Srp (green) alone. Embryos at this stage of development are marked by the appearance of intersegmental furrows (G, arrowheads). (I) Overlay is shown. Since Srp functions as a transcription factor, its localization is distinct from FKBP14 expression in hemocytes (inset). (J – L) A dorsal view of 16-18 h WT embryos shows FKBP14 staining (red) possibly in the fat body (arrowheads point to two regions) and in cells that migrate throughout the hemolymph that also express the collagen type IV protein, viking-GFP (vkg-GFP; green). An insertion in the viking locus causes expression of vkg-GFP fusion protein in hemocytes and the fat body. FKBP14 hemocyte localization is not detected in a snail-twist embryo (J, inset). (L) Overlay. GFP localization partially overlaps with FKBP14 (yellow; inset).

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interiorly located [406]. I detect FKBP14 localization in a subset of cells that express Serpent in

16 – 18 h embryos, likely hemocyte cells (Fig. 4.3 G – I). Plasmatocyte and fat body

localization is also observed with the collagen type IV factor known as viking [143], and

therefore I used viking-GFP fusion proteins to identify these structures in 16 – 18 h embryos. I

detect FKBP14 colocalization with viking-GFP in hemocytes, and observe possible faint

staining in the fat body at this focal plane (Fig. 4.3 J – L; arrowheads point to regions of the fat

body). FKBP14 hemocyte localization is no longer detected in the hemocyte-defective mutant

snail-twist/CyO (Fig. 4.3 J, inset). The exposure time was increased compared to other samples,

therefore FKBP14 staining in the gut, for instance, may be due to background detection.

4.4.3 FKBP14 is broadly expressed in third instar larval discs

I then wanted to examine FKBP14 expression in FKBP14D34 larval imaginal tissues. I

found that FKBP14 is expressed throughout third instar larval wing discs (Fig. 4.4 A), which

have been magnified to illustrate broad expression at the presumptive wing margin (Fig. 4.4 B)

and in the presumptive notum region (Fig. 4.4 C). I demonstrate that in eye discs, FKBP14

expression is detected in presumptive eye and antennal tissues (Fig. 4.4 D) and may accumulate

in photoreceptor cells (Fig. 4.4 E; arrows point to two putative photoreceptor cells). In larval

CNS, FKBP14 localizes to specific cells in the ventral nerve cord (Fig. 4.4 F; representative

cells have been magnified in Fig. 4.4 G) and in the neural lamina of third instar optic lobes (Fig.

4.4 H; magnified in Fig. 4.4 I).

4.4.4 FKBP14 overexpression increases Psn protein levels and affects Notch signalling

Since a loss of FKBP14 resulted in Psn-dependent Notch-related phenotypes and a

reduction in PsnNTF levels in pharate adults (Chapter 3), I wanted to examine the effects of

FKBP14 overexpression on Psn levels and Notch signalling in vivo. FKBP14 has two isoforms

known as FKBP14-PA (Fig. 4.5 A) and FKBP14-PB, which has an additional 15 N-terminal

residues (described in Chapter 3). I amplified cDNA of the shorter isoform, FKBP14-RA, to

generate FKBP14-Myc and FKBP14-V5 fusion proteins. Similarly, Psn encodes two isoforms

that differ in the cytoplasmic loop, as discussed [262], and the longer version, Psn-PA, was used

in this study to generate Psn-Myc and Psn-V5 fusion proteins (Fig. 4.5 A). In Kc cells, FKBP14

and PsnCTF are endogenously expressed and partially colocalize (Fig. 4.5 B - D), although in S2

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Figure 4.4 FKBP14 is broadly expressed in larval imaginal discs. (A) In FKBP14D34 third instar larval wing discs, FKBP14 staining appears ubiquitous, as further demonstrated in magnified (B) wing pouch and (C) presumptive notum regions. (D) FKBP14 is broadly expressed in FKBP14D34 imaginal eye discs, and may (E) localize to putative photoreceptor cells (arrows point to two examples). (F) FKBP14 localizes to cells of the ventral nerve cord in FKBP14D34 larval CNS, magnified in (G), and (H) to cells of third instar larval optic lobe anlagen, magnified in (I). Bars, 10 uM.

cells, levels of PsnCTF and PsnNTF are below detection by immunocytochemistry (personal

observation). Since an interaction between FKBP14 and Psn holoprotein was observed in S2

cells, as discussed in Chapter 3, I analyzed FKBP14 overexpression in S2 cells. I found that

constitutive expression of FKBP14-Myc or FKBP14-V5 in S2 cells for 48 h resulted in a 20%

increase in PsnNTF levels, compared to controls (Fig. 4.5 E). Psn holoprotein was only detected

when Psn-Myc and Psn-V5 were overexpressed (Fig. 4.5 E).

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Figure 4.5 FKBP14 overexpression increases Psn protein levels in S2 cells. (A) FKBP14 and Psn fusion proteins were engineered to express C-terminal Myc and V5 epitope tags (red). FKBP14-PA corresponds to a shorter FKBP14 isoform and Psn-PA denotes a longer Psn isoform. (B – D) Endogenous FKBP14 (red) and PsnCTF (green) partially overlap (yellow; merge), in Kc cells. DAPI is shown in blue. (E) S2 cells were transfected with LacZ, Psn-Myc, Psn-V5, FKBP14-Myc and FKBP14-V5 constructs cloned in the pAc5.1/V5-His plasmid for constitutive expression. FKBP14-tagged overexpression results in a 20% increase in PsnNTF levels compared to Psn-tagged and LacZ overexpression controls and an untransfected (Unt) control (top blot). Psn holoprotein (PsnFL-tagged), aggregates, endogenous Psn N-terminal fragments (PsnNTF) and unspecified cleavage bands are detected in Psn-tagged overexpression extracts (top blot). FKBP14 antibodies detect endogenous FKBP14 in addition to tagged fusion proteins (FKBP14-tagged; middle blot). β-tubulin was used as a loading control (bottom blot).

To determine if C-terminal tags affect FKBP14 and Psn subcellular localization, I

analyzed the expression of FKBP14 and Psn proteins engineered to express C-terminal V5 tags

in Kc cell culture. My analysis demonstrates that endogenous FKBP14 and constitutively

expressed FKBP14-V5 proteins localize to the same compartment (Fig. 4.6 A – C). Similarly,

Psn and Psn-V5 proteins localize to similar compartments (Fig. 4.6 D – F). FKBP14-Myc and

Psn-Myc proteins exhibit similar localization in S2 cells (data not shown). These data also

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Figure 4.6 FKBP14 and Psn proteins that express C-terminal tags exhibit similar subcellular localization as endogenous proteins. (A) Endogenous FKBP14 (red) and FKBP14-V5 proteins (green) colocalize (yellow; merge) in Kc cells. (B) Endogenous PsnNTF (red) and Psn-V5 (green) proteins overlap (yellow; merge) in Kc cells. DAPI is shown in blue.

resemble the subcellular localization of endogenous FKBP14 and Psn proteins shown in Fig. 4.5

B – D, suggesting that tagged proteins localize to endogenous compartments in vivo.

Since constitutive expression of FKBP14 in S2 cells increased PsnNTF levels, I wanted to

examine the effects of FKBP14 overexpression on Psn levels in vivo. UAS-FKBP14-Myc and

UAS-Psn-Myc constructs, described in Materials and Methods, were used in GAL4/UAS assays

as shown in Fig. 4.7. Importantly, FKBP14-Myc and Psn-Myc co-overexpression using the

tubulin-GAL4 driver (FM2/PM9; tub) in pupae raised at 29°C resulted in up to 85% increase in

Psn holoprotein (PsnFL) levels and up to 80% increase in PsnNTF levels, compared to controls

(Fig. 4.7, left side). I also detected up to 50% increase in PsnNTF levels when FKBP14 alone is

overexpressed using the tubulin-GAL4 driver (tub; FM3) in adult flies (Fig. 4.7, right side).

I then wanted to determine the effects of FKBP14 and Psn overexpression in vivo. Using

the GAL4/UAS system of targeted expression, I overexpressed UAS-FKBP14-Myc and UAS-

Psn-Myc transgenes in larvae and pupae raised at 29°C. This resulted in adult bristle

phenotypes in the notum (Fig. 4.8 A – C). Quantitation of these phenotypes is shown in Fig. 4.8

D – F. FKBP14-Myc overexpression resulted in a mild bristle phenotype (Fig. 4.8 A – C; left

side panels), using tub-GAL4 and pnr-GAL4 to drive expression. A statistically significant

increase in bristle numbers is observed when pnr-GAL4 drives FKBP14-Myc expression,

compared to controls (Fig. 4.8 D; n = 24; **p£0.01). Similarly, Psn-Myc overexpression by

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Figure 4.7 FKBP14 overexpression increases Psn protein levels in vivo. UAS-Psn-Myc and UAS-FKBP14-Myc transgenes were co-overexpressed using tubulin-GAL4 (FM2/PM9; tub) in 24 h pupae raised at 29°C. This resulted in up to 85% increase in PsnFL levels and up to 80% increase in PsnNTF levels, compared to the following controls: tub, PM9, FM2, FM2/EGFP; tub, PM9/EGFP; tub, PM9; tub, FM2; tub and EGFP; tub (top blot, left). In adult flies, FKBP14 overexpression (tub/FM3) results in up to 50% increase in PsnNTF levels compared to tub, PM5, FM3 and tub/PM5 controls (top blot, right). Psn overexpression in vivo also results in increased holoprotein, aggregates and cleavage products without increased PsnNTF levels (top blot, right). Endogenous FKBP14 and FKBP14-Myc fusion proteins are detected with an anti-FKBP14 antibody (middle blot). β-tubulin was used as a loading control (bottom blot). Abbreviations are as follows: tubulin-GAL4 (tub), UAS-Presenilin-Myc (PM5, PM9), UAS-FKBP14-Myc (FM2, FM3) and UAS-EGFP (EGFP). Quantitation was performed as described in Materials and Methods.

tub-GAL4 (Fig. 4.8 A – C; left side panels) and pnr-GAL4 (data not shown) resulted in

supernumerary adult notal bristles at levels that are highly significant compared to controls (Fig.

4.8 D; n = 24; **p£0.01, ***p£0.0001). Since FKBP14 overexpression phenocopies what is

observed with Psn overexpression, I wanted to examine the effects of FKBP14 and Psn co-

overexpression. I observed a synergistic increase in adult notal bristles when FKBP14 and Psn

are co-overexpressed using tub-GAL4, compared to controls (Fig. 4.8 A – C; middle panels).

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Figure 4.8 Quantitation of FKBP14 and Psn overexpression Notch-related bristle phenotypes. (A - C) Schematics of adult nota (adapted from [119, 410]) are displayed beside panels of nota to the right that correspond to each diagram. Dots mark positions of adult bristles, and boxes highlight regions that have been magnified in the panels to the right. The bristle phenotypes are representative for each corresponding genotype. (A – C, left hand side) FKBP14 overexpression (pnr/FM3 and tub/FM3) results in up to 2 extra notal bristles. Similarly, Psn overexpression (PM9; tub) results in up to 3 extra notal bristles. (A – C, middle) Co-overexpression of FKBP14 and Psn transgenes (FM2/PM9; tub) results in a synergistic bristle phenotype in adult male flies compared to FM2/EGFP; tub and PM9/EGFP; tub controls. (A – C, right hand side) Overexpression of a strong Psn transgene (pnr/PM5) in an FKBPD34 (D34) genetic background, results in up to 11 extra scutellar bristles in adult male flies. This phenotype is suppressed in FKBP14D58 (D58) adult males, which display 6 extra scutellar bristles. (D – F) Quantitation of bristle phenotypes. (D) A significant increase in bristle numbers is observed in adult flies from pnr/FM3, PM9; pnr and PM9; tub overexpression lines, but not in the tub/FM3 line, compared to pnr, tub, FM3 and PM9 controls (n = 24; **p£0.01, ***p£0.0001). (E) An increase in bristle numbers observed in FM2/PM9; tub co-overexpression male adult flies is significant compared to FM2; tub, EGFP; tub, FM2/EGFP; tub and PM9/EGFP; tub controls (n = 20; **p£0.01). The PM9/EGFP; tub line displays a weaker bristle phenotype compared to PM9; tub, due to dilution of GAL4. (F) A decrease in bristle numbers in D58; pnr/PM5 transheterozygous 24 h pupae is highly significant compared to pnr, PM5, D34; pnr, D58; pnr, pnr/PM5 and D34; pnr/PM5 controls (n 40; ***p£0.0001). Abbreviations: FKBP14D34 (D34), FKBP14D58 (D58), pannier-GAL4 (pnr), tubulin-GAL4 (tub), UAS-Presenilin-Myc (PM5, PM9), UAS-FKBP14-Myc (FM2, FM3) and UAS-EGFP (EGFP). An unpaired Student’s t-test was performed for all statistical analyses.

This result is statistically significant (Fig. 4.8 E; n = 20; **p £ 0.01). In comparison, I observed

reduced levels of adult notal bristles in FKBP14D58; pnr-Psn flies raised at 29°C, compared to

FKBP14D34; pnr-Psn and controls (Fig. 4.8 A – C; right side panels). This is statistically

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significant (Fig. 4.8 F; n 40; ***p£0.0001), and is consistent with what was observed in our

genetic screen, as discussed in Chapter 2.

4.5 Discussion

FKBP14 is highly conserved in Metazoa, illustrating a requirement for FKBP14 in both

simple and complex systems. I demonstrate that FKBP14 is expressed throughout development

in Drosophila. My analysis also shows that Drosophila FKBP14 has a maternal contribution. I

show that FKBP14 is expressed in egg chambers, embryonic hemocytes and within larval

ventral nerve cords. I further demonstrate that FKBP14 overexpression resulted in a significant

increase in Psn protein levels. Additionally, I show that FKBP14 overexpression defects

phenocopy Psn-dependent Notch-related bristle defects. Finally, I determined that FKBP14 and

Psn co-overexpression resulted in synergistic bristle phenotyes of adult nota, suggesting that

both proteins function within the same pathway.

In Drosophila, maternal proteins regulate early embryonic patterning up to 2 – 3 hours

after egg laying, at which time zygotic expression of genes is activated, concomitant with some

maternal transcript degradation [411]. After this stage, the embryo develops under control of

the zygotic genome. I determined that FKBP14 proteins are maternally expressed, and can be

detected in 0 – 3 h embryos, suggesting that FKBP14 protein is required for early patterning of

WT embryos. I further demonstrated that FKBP14 levels increase 6 h after egg laying,

consistent with FKBP14 in situ data [412]. My analysis determined that FKBP14 expression is

stable throughout WT larval and pupal development, when key signalling events required for

sense organ determination are occurring. Since FKBP14 has a maternal contribution, detection

of FKBP14 proteins in FKBP14 null embryonic, larval and pupal extracts suggests that these

may be maternal proteins. As expected, I observed significantly reduced levels of FKBP14

proteins in FKBP14 mutant larval and pupal extracts compared to WT.

In preblastoderm embryos, maternal transcripts and proteins can be detected throughout

the cytoplasm, prior to cellularization [413]. At this stage, I observed FKBP14 expression in the

cytoplasm that surrounds nuclei. After 3 h of embryonic development, germ band elongation

begins, and will terminate at approximately 6 h after egg laying, or at stage 11 according to the

Bownes stages of embryo development [408, 409]. At this stage, pro-hemocytes, which are

precursors to immune cells, are detected in cephalic mesoderm and throughout the hemolymph

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[139]. The GATA transcription factor known as Serpent localizes to pro-hemocytes and midgut

[406], and I demonstrate that FKBP14 is expressed in a subset of Serpent-positive pro-

hemocytes and in the midgut.

Hemocytes comprise three specialized Drosophila immune cells that differentiate from

pro-hemocytes during stages 11 – 17. These cells are involved in phagocytosis (plasmatocytes),

melanization (crystal cell) and encapsulation of parasites (lamellocytes) [139, 140, 142].

Hemocytes are also involved in distributing extracellular matrix components of basement

membranes that surround muscle and fat cells and the ventral nerve cord [362, 363]. Previous

studies show a requirement for serpent and the collagen type IV factor viking in hemocyte

development [143, 414]. In 16 – 18 h embryos, I demonstrate that FKBP14 localized to cells

that also express Serpent and viking. To confirm FKBP14 hemocyte localization, I examined

FKBP14 expression in the hemocyte-deficient mutant known as snail-twist and as expected, I

did not detect hemocyte-specific expression of FKBP14. Altogether, these data suggest that

FKBP14 may play a role in embryonic hematopoiesis.

I then determined FKBP14 expression in larval imaginal tissues. Imaginal discs comprise

thin epithelial cell layers that respond to multiple signalling pathways during larval and pupal

development to specify the corresponding adult structures [415]. I found that FKBP14 is

broadly expressed in wing and eye discs, although there may be enhanced localization in

photoreceptor cells. In larval CNS, FKBP14 localizes to specific cells in the ventral nerve cord.

In the developing visual system, retinal axons project to stereotypical positions within the neural

lamina of each optic lobe [416, 417]. I also observed FKBP14 localization in the neural lamina,

suggesting that FKBP14 expression is required in developing optic lobes.

A loss of FKBP14 function results in bristle defects and reduced PsnNTF levels, as

discussed in Chapter 3. If FKBP14 function is required for Psn protein maintenance, I would

expect that overexpression of FKBP14 may disrupt Psn protein levels as well. I therefore

wanted to determine the effects of FKBP14 overexpression on Psn levels and the Notch

pathway. My analyses demonstrated that endogenous FKBP14 and PsnCTF proteins partially

colocalized in the ER in Kc cells. Additionally, constitutive expression of FKBP14-tagged

proteins in S2 cells resulted in a 20% increase in PsnNTF levels, compared to controls. A lack of

Psn holoprotein in FKBP14 overexpression extracts suggests that FKBP14 stabilizes steady-

state Psn levels, and may not be involved in upregulation of Psn protein.

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Since constitutive expression of FKBP14 in S2 cells increased PsnNTF levels, I wanted to

examine the effects of FKBP14 overexpression on Psn levels in vivo. I detected up to 50%

increase in PsnNTF levels when FKBP14 is expressed in adult flies. I further demonstrated that

FKBP14 and Psn co-overexpression in pupae resulted in up to 85% increase in Psn holoprotein

and up to 80% increase in PsnNTF levels compared to controls. These data confirm that FKBP14

is specifically required to maintain Psn levels in vivo.

To examine the developmental defects that result from FKBP14 and Psn overexpression in

vivo, I overexpressed both proteins using the GAL4/UAS system of targeted expression. I

demonstrate a statistically significant increase in numbers of adult bristles when FKBP14-Myc

and Psn-Myc transgenes are expressed using the pannier-GAL4 driver (n = 24; **p£0.01 or

***p£0.0001). These results show that FKBP14 overexpression phenocopies what is observed

with Psn overexpression, suggesting that both proteins may be involved during bristle

development. I therefore examined the effects of FKBP14 and Psn co-overexpression, and

observed synergy of adult notal bristle phenotypes when FKBP14-Myc and Psn-Myc are co-

overexpressed using tub-GAL4. This result is statistically significant (n = 20; **p £ 0.01). My

analysis also demonstrated reduced levels of adult notal bristles in FKBP14D58; pnr-PM5 flies

compared to FKBP14D34; pnr-PM5, which is statistically significant (n 40; ***p £0.0001).

Together, these data suggest that FKBP14 and Psn activities are required within the same

pathway.

Altogether, my analyses demonstrate that FKBP14 is broadly expressed throughout

Drosophila development. Additionally, I confirmed a requirement for FKBP14 in Psn protein

maintenance, and determined that both proteins are involved during bristle development. I will

next address whether FKBP14 affects other components of the γ–secretase complex.


Serpent antibodies were provided by Dr. D. Hoshizaki. The pAc5.1/V5-His expression

vector was provided by Dr. Julie Brill.

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CHAPTER 5

EFFECTS OF THE IMMUNOSUPPRESSANT DRUG FK506 ON THE GAMMA-

SECRETASE COMPLEX

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5.1 Summary

Assembly of the four core components of the g-secretase complex, known as PSEN,

PSENEN, APH1 and NCSTN in vertebrates, within the early secretory pathway is highly

regulated, and may involve additional cofactors, including CD147, Rer1 and TMP21.

Activation of the complex is believed to occur downstream of the ER, and an active g-secretase

complex then catalyzes the final intramembrane cleavage of substrates in late endosomal

compartments or at the cell surface. I previously determined that Drosophila FKBP14 and

PsnCTF endogenously colocalized in the ER. Here, I demonstrate that endogenous FKBP14

colocalizes with Aph-1 in the ER. I also show that FKBP inhibition by the immunosuppressant

factor, FK506, results in significantly reduced levels of Aph-1 and Pen-2 and a mild reduction in

PsnNTF levels. Altogether, these data suggest that FKBP14 may be involved in early maturation

of the g-secretase complex in flies.

5.2 Introduction

The g-secretase complex comprises four essential proteins, known as anterior pharynx

defective 1 (APH1), presenilin enhancer 2 (PSENEN), nicastrin (NCSTN) and Presenilin in

vertebrates. This complex activates the final intramembrane cleavage of more than 30 known

type 1 transmembrane domain proteins, including Notch and APP [8, 54]. The precise role of

each component is currently unknown, although numerous studies have implicated a presenilin

heterodimer as the catalytic core of the active complex. Nicastrin may be required for

intracellular trafficking of the complex and for substrate recognition at the cell surface [44, 47-

49], whereas anterior pharynx defective 1 has been implicated in complex stability [50]. After

or perhaps during endoproteolytic cleavage, presenilin N- and C-terminal fragments are

stabilized via an interaction with the C-terminus of presenilin enhancer 2 [28, 51-53].

Numerous studies have shown that γ-secretase is predominantly localized within ER-Golgi

compartments [41, 50, 65-67], although small amounts have been observed at the cell surface

[27, 68]. The formation of an early subcomplex between anterior pharynx defective 1 and

nicastrin within the ER is believed to be the first step in stabilizing Presenilin holoprotein [28,

66, 67]. The remaining steps involve incorporation of PSEN and PSENEN into an APH1-

NCSTN subcomplex in the ER, and trafficking to the Golgi for complex activation [41, 65, 66],

similar to what has been observed in Drosophila [28, 50]. However, complex assembly may

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also occur within post-ER compartments, such as the intermediate compartment and cis- and

trans-Golgi networks [42, 69], and may require other factors for transport, stability and

maturation. For instance, Retrieval to the Endoplasmic Reticulum 1 (Rer1) interacts with

NCSTN [42] and PSENEN [70] in the intermediate compartment or cis-Golgi, to facilitate

retrieval of unassembled components to the ER. Presenilin enrichment has also been observed

in COPI-coated vesicles, which traffic between ER and Golgi compartments, suggesting that

there may be additional retrieval molecules involved in complex assembly [24, 54]. Additional

γ-secretase cofactors suggests that assembly and trafficking of the complex is highly regulated.

Here, I demonstrate that FKBP14 colocalizes with Drosophila Aph-1 in the ER of S2 cells,

suggesting that FKBP14 may be involved in Aph-1 regulation. Since FKBP14 colocalizes and

binds with Psn in the ER and is required for Psn protein maintenance (described in Chapter 3),

this implicates FKBP14 function in Aph-1 protein regulation.

To assess the role of FKBP14 in complex assembly, I examined the effects of an

immunosuppressant drug, known as FK506, on components of the γ-secretase. FK506

(Tacrolimus, Prograf) reduces the immune response and lowers the risk of organ rejection in

human transplant patients [218-220]. FK506 binds to the PPIase domain of cytosolic FKBP12,

inhibiting its PPIase activity, and subsequently other FKBPs have displayed varying affinities

for the drug [182, 186, 223, 224]. The affinity between FKBP14 and FK506 has yet to be

determined. I transiently expressed Drosophila Aph-1, Pen-2 and Nct constructs in S2 cells,

which endogenously express FKBP14 and Psn proteins. My analysis demonstrates that FK506

treatment resulted in a significant reduction in Aph-1, Pen-2 and Psn protein levels after 24

hours and had no detectable effect on Nct levels. Altogether, my data demonstrate a putative

interaction between FKBP14 and Aph-1 in the ER, and supports a model whereby FKBP

activity may be required for complex assembly and stability.


5.2.1 Plasmids

Aph-1-FLAG, HA-Pen-2 and Nct-V5 plasmids were a gift of Dr. Iwatsubo [28].

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Extracts for Western analysis were prepared using standard procedures. S2 cells harvested

48 h post-transfection were homogenized in RIPA buffer (1% NP40, 0.5% sodium

deoxycholate, 0.1% SDS, 0.02 molar Tris-Cl pH 8.0, 0.137 molar NaCl, 10% glycerol, 2 uM

EDTA) supplemented with protease inhibitors (Roche). Lysates were centrifuged at 10,000g for

15 minutes and supernatants were analyzed. SDS-polyacrylamide gel electrophoresis (10%

SDS-PAGE) and immunoblot analyses were carried out as described [262].

The following primary antibodies were used: rabbit a-PsnNTF [56], rat a-FKBP14

(Chapter 3; 1:100), mouse a-FLAG detects Aph-1-FLAG proteins (Sigma; 1:2000) mouse a-

HA detects HA-Pen-2 proteins (Roche; 1:1000) and mouse a-V5 detects Nct-V5 proteins

(Invitrogen; 1:5000). Experimental input was determined using mouse anti-b-tubulin from the

Developmental Studies Hybridoma Bank. HRP secondary antibodies were used (Jackson

ImmunoResearch; 1:10 000). All blots were performed in triplicate. Quantitation was

performed using a Fluorchem 8000 Gel Documentation System and Alpha Innotech software

(San Leandro, CA).

5.2.3 Immunostaining

Immunostaining was performed using standard procedures [377-379]. S2 cells were fixed

at RT in 4% paraformaldehyde for 10 min, washed in PBS, permeabilized in 0.1% Triton and

PBS (PBT) and non-specific interactions were blocked in 5% normal donkey serum (NDS)

diluted in PBT for 1 hour. Cells were then incubated with primary antibodies diluted in PBT

containing 5% NDS for 1 hour at RT, washed in PBT followed by incubation with secondary

antibodies in PBT for 1 hour at RT. After 3 washes in PBT, samples were mounted in Dako

(DakoCytomation).

The primary antibodies: rat a-FKBP14 (1:100), mouse a-FLAG (Sigma; 1:200), mouse a-

HA (Roche; 1:200) and mouse a-V5 (Invitrogen; 1:200), and A488 and Cy3 secondary

antibodies (Jackson ImmunoResearch; 1:1000) were used. DAPI was used at 1:5000. Images

were obtained using a Leica DMRA2 Fluorescence Microscope and processed in Photoshop CS

and Illustrator CS.

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5.2.4 Cell culture

S2 cells were maintained at RT in Schneider’s media supplemented with 10% fetal bovine

serum. Cells were transfected with 3 ug of plasmid DNA using Cellfectin (Invitrogen). After

supplementation of 30% fetal bovine serum, transfected cells were treated for 24 hours (24 h)

with 50 uM FK506 (Prograf; The Hospital for Sick Children Pharmacy) and the DMSO vehicle

control, at RT.

5.3 Results and Discussion

5.3.1 FKBP14 and Aph-1 colocalize in the ER

The g-secretase complex comprises four core proteins, known as Aph-1, Pen-2, Nct and

Psn in flies [28, 31]. This complex is highly regulated, requiring the interplay of each

component for stable assembly within the ER compartment [28]. Reduced levels of individual

g-secretase components affects Psn endoproteolysis, a key step in g-secretase maturation that is

required for activity [28, 35]. Initially, a subcomplex in the ER comprising anterior pharynx

defective 1 and nicastrin is believed to target Psn holoprotein [41, 66, 405]. Pen-2 is required

for Psn holoprotein stability [28] and is incorporated into the complex prior to exiting from the

ER [41, 405]. Presenilin endoproteolysis then occurs downstream of the ER [405] generating C-

and N-terminal fragments which are required for g-secretase activity [32]. An active g-secretase

complex catalyzes the final intramembrane cleavage of APP and Notch [8]. Other factors

involved in g-secretase assembly or stability have been identified, including CD147, Rer1 and

TMP21 [70-72]. I observed an interaction between FKBP14 and Psn, which is required to

maintain Psn N-terminal fragment (PsnNTF) protein levels (Chapter 3). Here, I wanted to assess

whether FKBP14 regulates other components of the g-secretase complex. To do this, I

examined the subcellular localization of the g-secretase components, using epitope tagged Aph-

1, Nct and Pen-2 constructs. I demonstrate that endogenous FKBP14 partially colocalizes with

Aph-1-FLAG proteins (Fig. 5.1 A; right panels), putatively in the ER since FKBP14 localizes to

the ER (Chapter 3). My analysis also shows that Nct-V5 localizes to specific intracellular

compartments that are distinct from endogenous FKBP14 (Fig. 5.1 B; right panels), similar to

HA-Pen-2 localization (Fig. 5.1 C; right panels). These data suggest that FKBP14 may interact

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Figure 5.1 FKBP14 partially colocalizes with Aph-1 in vivo. (A) Endogenous FKBP14 (red) and Aph-1-FLAG (Aph-1; green) partially colocalize in transfected S2 cells, shown in yellow in the merge. (B) Endogenous FKBP14 (red) and Nct-V5 (Nct; green) localize to separate intracellular compartments, indicated by a lack of yellow in the merge. (C) Similarly, endogenous FKBP14 (red) and HA-Pen-2 (Pen-2; green) localize to separate intracellular compartments (merge). DAPI is shown in blue. Protein structures of the g-secretase components are shown in Chapter 1, Fig. 1.1.

with Aph-1, possibly in early subcomplex formation. Alternatively, FKBP14 may be involved

in g-secretase- independent Aph-1 activity.

5.3.2 FK506 decreases Aph-1, Pen-2 and Psn protein levels in S2 cells

To determine whether FKBP14 is involved in regulating the γ-secretase complex, I tested

the effects of an FKBP inhibitor, known as FK506, on levels of ectopically expressed Aph-1,

Pen-2 and Nct constructs and on endogenous Psn levels. FK506 is a naturally occurring

macrolide that was discovered over 20 years ago [219], and is commonly administered to organ

transplant patients to inhibit calcineurin activation of T lymphocytes, thereby preventing organ

graft rejection [219, 418, 419]. FKBP12 was identified as the original FK506 binding target

[187], and subsequently other FKBPs have been observed in FK506/FKBP complexes, although

105

with varying affinities [219, 224, 226]. I show that after 24 hours of 50 uM FK506 treatment in

transfected S2 cells, levels of Aph-1-FLAG and HA-Pen-2 are significantly reduced (Fig. 5.2 A

and B). I also observed a decrease in endogenous PsnNTF levels after 24 h treatment (Fig. 5.2

B). Since FKBP14 is endogenously expressed in S2 cells (described in Chapter 3), these data

suggest that FKBP14 function may be involved in regulating multiple g-secretase components.

Nct-V5 levels are stable throughout treatment (Fig. 5.2 C), indicating that FKBP14 may not be

required to maintain Nct proteins. In the control experiment, involving treatment with DMSO

alone, I found that levels of all four g-secretase components are stable.

A major caveat to these results is that multiple FKBPs may be targeted by FK506 [219,

226]. For instance, there are 8 currently known mammalian FKBPs that form a complex with

FK506, including FKBP12, FKBP12.6, FKBP13, FKBP25, FKBP38, FKBP51, FKBP52 and

FKBP65 [219, 226], which exhibit varying affinities for the drug [219]. Since Drosophila

encode 8 FKBPs, known as FKBP14, FK506-bp2 (FKBP12), FK506-bp1 (FKBP39), FKBP59,

shutdown, CG14715, CG5482 and CG1847, this suggests that FK506 may disrupt multiple

FKBP functions. Additionally, the number of FKBPs expressed in S2 cells, which were derived

from late-stage embryos and exhibit hemocyte- like gene expression [420], is currently unknown.

However, data from a Drosophila in situ database demonstrates that 3 FKBPs, including

FKBP14, FKBP59 and CG1847, localize to hemocytes [412]. I also demonstrated endogenous

FKBP14 protein expression in S2 cells (Chapter 3). Together with my colocalization data, these

results suggest that FKBP14 may be involved in Psn, Pen-2 and Aph-1 protein maintenance.

Furthermore, these data implicate FKBP14 activity in g-secretase complex assembly within

early secretory compartments.

5.4 Conclusions

Psn, Aph-1, Pen-2 and Nct constitute the core components of the g-secretase complex [28,

31]. I have demonstrated that FKBP14 partially colocalized with Aph-1 in S2 cells. My

analysis has also shown that inhibition of FKBP activity in S2 cells, by the immunosuppressant

drug FK506, resulted in decreased Aph-1, Pen-2 and PsnNTF protein levels, whereas Nct levels

appeared stable. This suggests that FKBPs that localize to S2 cells, and bind FK506, may be

involved in γ-secretase regulation. I previously observed partial colocalization between

FKBP14 and Psn in Kc cells, and determined that FKBP14 is required to maintain Psn protein

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Figure 5.2 FK506 treatment disrupts Aph-1 and Pen-2 overexpression levels, and endogenous PsnNTF protein levels. (A) Aph-1-FLAG levels are reduced after 24 h treatment of 50 uM FK506. (B) HA-Pen-2 levels are significantly reduced between 6 – 24 hour treatment of 50 uM FK506. PsnNTF levels are decreased after 24 h treatment of FK506. (C) Nct-V5 levels are unaltered by FK506. DMSO has no effect on γ-secretase protein levels. β-tubulin was used as a loading control. These blots are representative of three individual experiments.

levels in vivo (Chapter 3). These data suggest that FKBP14 may be involved in early g-secretase

complex assembly. Since FKBP14 orthologs in yeast exhibit chaperone activity, colocalization

between Drosophila FKBP14 and Psn or Aph-1 implicates FKBP14 function in γ-secretase

regulation. Further analysis of the role of FKBP14 in γ-secretase complex assembly will be

discussed in Chapter 6.


The pAph-1-FLAG, pNct-V5 and pHA-Pen-2 expression vectors were provided by Dr. T.

Iwatsubo [28].

107

CHAPTER 6

PROSPECTIVES AND FUTURE DIRECTIONS

108

6.1 Summary

I have described the identification of additional putative Presenilin- interacting genes, and

determined that one of these genes, FKBP14, is involved in Presenilin protein maintenance in

flies. I have also demonstrated that FKBP14 loss-of-function mutants are lethal, and show a

requirement for FKBP14 function during larval disc development. I determined that FKBP14

function may be involved in proneural gene regulation and Notch signalling activity.

Altogether, my analyses have identified a novel role of FKBP14 in Presenilin biology and

development, and have provided further insights into Presenilin activity.

6.2 Involvement of Psn in numerous intracellular activities

I have studied the role of Presenilin during development using a Drosophila Psn

dominant-negative system. My analysis has determined that Presenilin function may be

involved in numerous intracellular signalling pathways. In particular, key members of the

EGFR, FGF, Hh, Notch, Wnt/Wg and TGFβ/Dpp signalling pathways were isolated in a screen

for Psn- interacting genes, in addition to multiple intracellular calcium regulators and factors

involved in apoptosis regulation. It would be interesting to further examine the association

between Psn and its novel putative cofactors to define the nature of their protein-protein

interactions. In turn, it would be important to determine whether these Psn- interacting genes are

involved in Psn-associated AD pathology or in γ-secretase- independent Psn activity. Further

analysis is also required to determine whether the P-element alleles that were identified in our

screen confer a gain or a loss of gene function.

6.2.1 Examining the protein-protein interactions between Psn and the candidate genes

Presenilin was demonstrated to physically interact with the calcium signalling components

FKBP14 (Chapter 3) and RyR [148]. Since Psn genetically interacts with numerous other

factors identified in our screen, it would be interesting to further elucidate the association

between Psn and these candidate genes. In particular, it would be important to validate the

interactions, and then assess whether the proteins physically interact. To perform these

experiments, I propose to use null mutant alleles that are available from the Drosophila Stock

Centers, and to outcross these with the Psn null mutant, Psnw6rp, to confirm the interaction. A

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genetic interaction that is validated in this way could then be characterized further using an

immunoprecipitation approach to assess possible protein-protein interactions.

6.2.2 Examining the effects of Psn FAD-linked mutations on Psn modifier genes

FAD-linked mutations in presenilin associate with disruptions in ER calcium leak activity

[150], and enhanced apoptosis [154]. Therefore, I propose to analyze whether FAD mutations

affect the interaction between Psn and the calcium regulators, FKBP14 and RyR. Since I have

shown that Psn holoprotein and FKBP14 interact in cell culture, I would assess the ability of

endogenous FKBP14 and Psn FAD mutants to interact using an immunoprecipitation approach

in cells expressing FAD mutants. This would determine if FAD mutations inhibit the interaction

between Psn and FKBP14, and would provide further understanding of the possible role of

FKBP14 in Alzheimer’s disease. Similarly, reducing intracellular calcium flux with ER-specific

calcium pump inhibitors, such as thapsigargin [421], in Psn expressing cells would determine

whether calcium signalling is involved in the interaction between Psn and FKBP14. I would

then examine the effects of a loss of FKBP14 and Rya-r44F function in FAD-expressing

transgenic flies [263]. For instance, I would test the ability of FKBP14D58 and Rya-r44F16 null

alleles to disrupt the partial loss-of-function defects that are observed in FAD overexpression

flies [263]. Enhancement or suppression of these phenotypes would implicate FKBP14 or

Ryanodine receptor function in Psn-dependent AD pathology.

6.2.3 Examining a role for Psn in Drosophila hematopoiesis

Psn- interacting genes were identified that may be involved in hematopoiesis, including

for, gcm, Rab5, Ras85D, skd, smt3and tkv (discussed in Chapter 2). I also observed FKBP14

localization in hemocytes (discussed in Chapter 3). These data implicate a role for Psn in

Drosophila hemocyte development, although a specific requirement is currently unclear. Since

I observed endogenous Psn expression in Kc and S2 cells, which are believed to be derived from

embryonic hemocytes [420, 422, 423], this suggests a possible involvement of Psn in

hematopoiesis. Prohemocytes that originate from larval lymph glands express the GATA

transcription factor, Serpent. I propose to examine Serpent expression in larval lymph glands of

Psn mutant larvae to determine whether Psn plays a role in prohemocyte development. I would

expect a significant reduction in Serpent-positive cells in Psn null mutants if Psn function is

110

required during hematopoiesis. Additionally, a hemocyte-specific GAL4 driver, known as

Hemese-GAL4 (He-GAL4) [319], could be used to overexpress Psn. If Psn is involved in

hemocyte regulation, I would expect defects in the levels of hemocytes in larvae that

overexpress Psn. In this way, I can determine whether Psn function is involved during

hematopoiesis.

6.2.4 Characterizing the candidate gene mutations

The annotated fly genomic sequence and P-element mutational analyses have provided

critical information on the putative functions of the candidate genes in our genetic screen.

Analyzing whether a P-element mutation caused a loss or gain in gene expression will aid in

elucidating the effects on Psn overexpression that were observed in our genetic screen. To

address this, I suggest performing RT-PCR analysis for each P-element allele in order to

determine the profile of gene expression.

6.3 Examining the interaction between Psn and FKBP14

I have demonstrated a novel interaction between FKBP14 and Psn holoprotein in the ER

in cell culture. My analysis also determined that FKBP14 is involved in stabilizing Psn protein

within the ER. Since protein sequence data suggests that Drosophila FKBP14 encodes a PPIase

domain involved in protein folding, further analysis of this domain may determine if it is

involved in protein binding. FKBP14 orthologs in yeast have also been identified in protein

complexes that include other ER chaperones, and further analysis will determine whether the

interaction between FKBP14 and Psn in flies is direct or if it involves a complex of proteins.

6.3.1 Characterizing the site of interaction between Psn and FKBP14

FKBP14 orthologs in yeast, known as FKBP22, dimerize at the C-terminus and display

PPIase activity at the N-terminus [201]. Interactions between yeast FKBP22 and multiple

intracellular chaperones in the ER are mediated via the PPIase domain [201, 209]. Yeast

FKBP22 proteins share 33% sequence identity with Drosophila FKBP14 (Fig. 6.1), and display

highly conserved PPIase domains. These proteins also display similar secondary structures (Fig.

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6.1), based on a secondary structure prediction model [201, 424]. This secondary structure is

similar to what has been shown with human FKBP12, which displays a five-stranded

antiparallel β-sheet wrapped around a short a-helix [223]. Since the PPIase domains are highly

conserved, this region could be analyzed for the ability to bind to Psn.

To do this, I propose protein truncation analysis to identify the critical regions required for

binding. Generating Drosophila FKBP14 protein truncations and performing

immunoprecipitation assays in Psn-expressing cells would identify the critical regions that are

necessary and sufficient to mediate its interaction with Psn. Mutagenesis assays and subsequent

immunoprecipitation analysis could then be performed to identify the key binding residues. I

propose to mutate highly conserved residues located within the FKBP14 PPIase domain (Fig.

6.1), to examine whether this region is involved in mediating Psn protein binding and stability.

Similarly, if the interaction between Psn and FKBP14 was disrupted with FK506, which binds

to the PPIase domain of FKBP12 and inhibits its activity [182, 186, 223, 224], this would also

implicate the PPIase domain in protein binding.

6.3.2 Analysis of a direct interaction between FKBP14 and Psn in the ER

Psn has been shown to associate with many intracellular factors, including FKBP14

(Chapter 3). To address whether FKBP14 directly binds to Psn, I propose to perform a GST

fusion protein pull-down experiment. Specifically, I would use a GST-FKBP14 fusion protein

that was originally generated for antibody production, and assess its interaction with Psn using

purified recombinant Psn protein. Since highly conserved FKBP14 orthologs in yeast were also

shown to bind with multiple intracellular chaperones within the ER as part of chaperone

complexes [209], I propose to also examine whether Drosophila FKBP14 interacts with ER-

localized chaperones using a proteomics approach and mass spectrometry for candidate protein

analysis. Yeast lack a presenilin ortholog [31], therefore I suggest to also perform FKBP14

RNA-interference (RNAi) studies in mammalian cell culture to assess the role of FKBP14

function in vertebrates. This will determine whether FKBP14 orthologs are functionally

conserved. Interestingly, Neurospora crassa FKBP22 mutants exhibit a slow growth defect

[209] and Saccharomyces cerevisiae mutants lacking total FKBP functions are viable [425].

Thus, identifying additional FKBP14-binding partners in other species will facilitate our

understanding of the role that FKBP14 plays in vivo.

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Figure 6.1 A multiple sequence alignment of FKBP14 orthologs from fly and yeast. FKBP14 orthologs in fly (Dmel FKBP14, Accession NP_726074) and yeast (Neurospora crassa [Ncra] FKBP22, Accession O60046) display highly conserved residues (*), conserved substitutions (:) and semiconserved substitutions (.). The residues in Drosophila FKBP14 that comprise the PPIase domain are highlighted in bold, and correspond to residues 33 – 126 of the PPIase domain in yeast FKBP22. The critical residues comprising an FK506 binding pocket in FKBP12 [223] are highly conserved in FKBP14 orthologs (grey boxes). β-strands (red rectangles) and a-helices (blue rectangles) were predicted from the secondary structure prediction program Porter [424].

6.3.3 Examining the interaction between Psn and FKBPs

Flies encode 8 FKBPs, known as FK506-bp2 (FKBP12), FKBP14, FK506-bp1

(FKBP39), FKBP59, shutdown, CG14715, CG5482 and CG1847. These proteins exhibit weak

similarity, although FKBP12, FKBP13 and FKBP14 show high sequence similarity (Chapter 3).

In mammals, 15 FKBPs have been identified in the genome [184], and a specific FKBP, known

as FKBP38, was identified as a presenilin-binding factor in mammalian cell culture [154].

Since I demonstrated an interaction between Psn and FKBP14 in flies, this implicates a general

involvement of FKBPs in Psn protein binding. To assess whether Psn binds with other FKBP

family members in Drosophila, I propose to perform GST pull-down assays using purified Psn

protein and FKBP-GST fusion proteins immobilized on glutathione beads. In this way, the role

of FKBP function in Psn activity can be further elucidated.

6.4 Examining FKBP14 function in vivo

FKBP14 orthologs are conserved in Metazoa, illustrating a requirement in development.

In unicellular organisms, FKBP14 orthologs exhibit chaperone activities [209], in addition to

peptidyl-prolyl cis-trans isomerase activities that are required to catalyze the trans-to-cis

conversion of peptide bonds N-terminal of proline residues [209, 426]. I demonstrated that

Dmel-FKBP14 MNNFLEYISVLHCSSMSKSNLVISCLLLVAISNSLVRAQDLKVEVISTPEVCEQKSKNGD 60 Ncra-FKBP22 -----------------MKSIFLSLSLLASATVGVLAAEELGIDVT-VPVECDRKTRKGD 42 ..:.:* **.: : .:: *::* ::* .* *::*:::** Dmel-FKBP14 SLTMHYTGTLQADGKKFDSSFDRDQPFTFQLGAGQVIKGWDQGLLNMCVGEKRKLTIPPQ 120 Ncra-FKBP22 KINVHYRGTLQSNGQQFDASYDRGTPFSFKLGGGQVIKGWDEGLVDMCIGEKRTLTVPPS 102 .:.:** ****::*::**:*:**. **:*:**.********:**::**:****.**:**. Dmel-FKBP14 LGYGDQGAGNVIPPKATLLFDVELINIGNAP-PTTNVFKEIDDNADKQLS--REEVSEYL 177 Ncra-FKBP22 YGYGQRSIG-PIPAGSTLIFETELIGIDGVPKPESIVYKQAAEKAEEAASAVEEKVAEAT 161 ***::. * **. :**:*:.***.*...* * : *:*: ::*:: * .*:*:* Dmel-FKBP14 KKQMTAVEGQDSEELKNMLAENDKLVEEIFQHED--KDKNGFISHDEFSGPKHDEL 231 Ncra-FKBP22 DKAGGKIADATKKVEEKAEEASANVVEKVASVVSGAAEAVKTVVADTDDVQEHNEL 217 .* : . .: :: . ::**:: . . : : * . :*:**

113

Drosophila FKBP14 stabilized endogenous Psn protein in the ER, displayed a maternal

contribution, and was endogenously expressed in imaginal tissues. I determined that FKBP14

loss-of-function mutants were lethal, exhibiting defects in imaginal disc development possibly

due to misregulation of apoptosis, and adult phenotypes due to misregulation of proneural gene

expression and the Notch signalling pathway. This indicated that FKBP14 was essential during

development. Moreover, my analysis suggests that FKBP14 may be involved during embryonic

hematopoiesis. However, FKBP14 function in vivo remains to be further elucidated. Mainly, I

would like to determine whether FKBP14 is required for Psn protein stability during oogenesis

and hemocyte development or whether FKBP14 displays distinct intracellular roles.

6.4.1 Determining FKBP14 function in oogenesis

My analysis demonstrated an involvement of FKBP14 throughout oogenesis. FKBP14

localization was observed in the germarium and early-stage egg chambers, and in nurse cells,

oocytes and follicle cells of mid-stage and late-stage egg chambers. To further analyze the roles

of FKBP14 during oogenesis, I propose to generate FKBP14D58 germline clones using the FLP-

FRTovoD system. Initially, I would assess the effects of a loss of maternal and zygotic FKBP14

expression during embryogenesis, by analyzing whether FKBP14D58 germline clones lay eggs.

Alternatively, if FKBP14 is required during oogenesis, FKBP14D58 germline clones may exhibit

female sterility. Marker analysis could then be performed using oocyte-specific markers such as

anti-Orb [427], to determine whether FKBP14 is required for oocyte development, and an actin

cytoskeleton marker, Phalloidin, to assess the role of FKBP14 during egg chamber development.

Together, these analyses will help elucidate the requirement of FKBP14 during stages of

development when maternal FKBP14 proteins mask zygotic FKBP14 function.

6.4.2 Determining the role of FKBP14 in hematopoiesis

To assess the role of zygotic FKBP14 during embryogenesis, I propose to perform

FKBP14 RNA-mediated interference (RNAi). Previously, I performed FKBP14 double-

stranded RNAi experiments in cells derived from late-stage embryos, known as S2 cells. I

observed a mild reduction in FKBP14 protein levels after 6 days of treatment (data not shown).

This suggests that the dsRNAi technique may be only partially effective in cell culture.

Therefore, I propose to perform FKBP14 RNAi using the GAL4/UAS system of targeted

114

expression. Specifically, I would express UAS-FKBP14 RNAi transgenes, available from the

Vienna Drosophila RNAi Center (VDRC), using embryo-specific daughterless-GAL4 [428]

drivers to determine the effects of a loss of FKBP14 function during embryogenesis.

Additionally, I propose to overexpress FKBP14 transgenes using the hemocyte specific GAL4

driver, known as He-GAL4 [319], in developing embryos to determine the effect of FKBP14 on

hemocyte development. The He-GAL4 driver was originally used in a candidate genetic screen

to identify genes involved in Drosophila blood cell development. For instance, He-GAL4

overexpression of components of the EGFR and downstream Ras/MAPK pathways, including

Ras85D and EGFR, resulted in enhanced levels of hemocytes in larvae [319]. If FKBP14 is

required during hemocyte development, then overexpression of FKBP14 may disrupt hemocyte

levels. Together, these experiments will help elucidate whether FKBP14 function is essential

during hemocyte development.

6.4.3 Examining FKBP14 function in calcium homeostasis

FKBP14 exhibits an EF-hand calcium binding domain at the C-terminus, and it remains

unclear whether this motif is essential for FKBP14 function. I suggest generating point

mutations in the EF-hand motif to analyze its endogenous functions. Psn function has been

shown to be required for intracellular calcium signalling, and since Psn and FKBP14 interact, it

will be interesting to determine whether FKBP14 also plays a role in calcium regulation. To do

this, I would perform calcium flux experiments in S2 cells, using inhibitors of ER-specific

calcium pumps such as thapsigargin as described [421, 429, 430]. A store-operated calcium

entry pathway has been identified in S2 cells, and thapsigargin treatment was shown to deplete

intracellular calcium stores of S2 cells, using fluorescent calcium indicator molecules such as

Fluo-4, and a multiwell fluorescence plate reader as described [429]. A comparative analysis of

the effects of thapsigargin on calcium release could be similarly performed in S2 cells that

express endogenous FKBP14 versus S2 cells that express FKBP14 EF-hand mutants. If

FKBP14 is involved in intracellular calcium regulation, changes in Fluo-4 intensity that

correspond to changes in calcium release would be detected by fluorescence. This would

determine whether FKBP14 function, specifically its EF-hand motif, is involved in intracellular

calcium signalling.

115

6.4.4 Determining FKBP14 function in apoptosis

My analysis demonstrated a role of FKBP14 in mediating apoptosis in fly larvae. The

main defect in FKBP14 mutants involved a lack of discs, likely due to enhanced apoptosis at

earlier stages of development. I determined that FKBP14 mutant CNS and discs exhibited

enhanced apoptosis. I propose to further elucidate the role of FKBP14 function in apoptosis.

To do this, I would perform acridine orange immunostaining analysis on FKBP14D58 mutant

wing discs from early stages of larval development to determine at which stage the defects in

disc development are occuring. I also propose to perform co- immunoprecipitation and Mass-

Spectrometry analysis of WT flies to determine the protein targets of FKBP14 function. An

examination of the intracellular binding targets of FKBP14 would provide further insight into its

roles in vivo. Since FKBP14 mutants exhibit increased levels of apoptosis, I propose a co-IP

experiment aimed at identifying whether FKBP14 interacts with thread, a known anti-apoptotic

factor.

Psn proteins have also been shown to be cleaved by caspases as a response to intracellular

apoptosis, releasing smaller C-terminal fragments and larger N-terminal fragments compared to

the endogenous endoproteolytically cleaved fragments [173, 174]. Since I did not detect larger

PsnNTF fragments in FKBP14 mutant extracts using Western analysis (Chapter 3), it is unlikely

that Psn caspase cleavage occurs in these mutants. However, to determine whether Psn C-

terminal caspase cleavage products are produced in FKBP14 mutant tissues, I propose to

perform Western blot analysis using an anti-PsnCTF antibody that I described in Chapter 3. This

will further our understanding of the role of FKBP14 in Psn protein maintenance.

6.4.5 Is FKBP14 function involved in proneural gene activity?

FKBP14 function may be required for expression of the proneural gene, achaete, during

SOP development in presumptive nota. SOP levels are also significantly reduced in FKBP14

mutants, in addition to adult nota bristles. This was in contrast to what was observed in Psn null

mutants, suggesting that FKBP14 may play a role in proneural gene expression. Rescue

experiments aimed at restoring notum bristles in FKBP14D58 mutants, using UAS-achaete and

UAS-scute constructs [431, 432], would determine whether FKBP14 was involved in proneural

gene function. Since a loss of FKBP14 function resulted in reduced SOP levels and reduced

achaete expression, examining the interaction between FKBP14 and achaete will determine if

116

FKBP14 is directly involved in achaete protein maintenance. I suggest performing

immunoprecipitation analysis on endogenous proteins from third instar larval wing discs to

assess their ability to interact, using a monoclonal anti-achaete Ab and the anti-FKBP14

antibody which are both described in Chapter 3.

6.5 Analyzing the effects of FK506 on the γ-secretase complex

The traditional immunosuppressant drug, FK506, inhibits FKBP activities by competing

with key residues in a protein binding pocket [223]. Interestingly, I demonstrated significant

effects on the protein levels of components of the γ-secretase complex upon FK506 treatment

(described in Chapter 5). Clinical application of FK506 (Tacrolimus) in organ transplant

patients results in side-effects in some patients [218-220], although these may be ameliorated

using combination treatments [218, 220]. A portion of these effects may be due to reduced

FKBP14 activity, and subsequent defects in Psn protein stability which would disrupt signalling

pathways that involve numerous γ-secretase substrates. Determining whether FK506 treatment

affects FKBPs that are involved in Psn biology will further elucidate the efficacy of FK506 as

an immunosuppressant drug.

6.5.1 Determining FKBP expression in S2 cells

Characterization of the expression patterns of currently known ESTs in the Drosophila

genome has been compiled in a Drosophila in situ database [412]. As previously indicated, 8

FKBPs have been identified [57], and the expression patterns for 6 of these genes can be found

in the in situ database [412]. For instance, the expression pattern for shutdown revealed pole

cell and germ cell gene expression in embryos, and FKBP12 expression was detected in cephalic

mesoderm where hemocytes are believed to originate [139, 412]. Similarly, CG14715 gene

expression was detected in the lymph gland, fat body and midgut [412]. Three FKBPs exhibited

hemocyte-specific expression patterns, which include Fkbp13 (here described as FKBP14),

FKBP59 and CG1847 [412]. I also demonstrated endogenous FKBP14 expression in embryonic

hemocytes that express the GATA transcription factor Serpent and the collagen type IV

molecule, viking (Chapter 4). To further examine the expression of Drosophila FKBPs in cell

culture, I suggest performing RT-PCR analysis of gene expression in S2 cell extracts.

Additionally, I propose to analyze whether FKBP12 and shutdown proteins are expressed in S2

117

cells using immunocytochemistry with anti-FKBP12 [433] and anti-shutdown [434] antibodies.

In this way, the expression patterns of FKBPs in S2 cell culture can be assessed.

6.5.2 Examining the effects of FK506 inhibition of FKBPs in cell culture on γ-secretase

I have demonstrated that FK506 treatment in S2 cell culture reduces protein levels of the

γ-secretase components, Aph-1, Pen-2 and Psn (Chapter 5). Presenilin activity has also been

shown to be required for maintaining Pen-2 stability and for subcellular trafficking of Pen-2 in

mammalian cell culture [435]. FK506 targeting of FK506-binding proteins (FKBPs) inhibits

their PPIase activities, although the affinity for the drug varies amongst family members [224].

Together, these data suggest that FKBPs may modulate Psn function in vivo. Characterizing

FKBP expression in S2 cells, and detemining whether they exhibit an affinity for FK506, will

aid in our understanding of the role of FKBP function in γ-secretase protein stability. The

hemocyte-specific expression patterns observed for FKBP14, FKBP59 and CG1847, and the

effects of the immunosuppressant drug FK506 in S2 cells, indicates that these FKBPs may be

involved in mediating γ-secretase levels. Therefore, I propose to further analyze their

expression in cell culture to gain insights into their physiological roles. Performing RNAi

experiments on endogenous FKBP14, FKBP59 and CG1847 transcripts in cells expressing Aph-

1, Nct and Pen-2 transgenes, and determining the effects on Aph-1, Nct, Pen-2 and endogenous

Psn protein expression levels, will help elucidate the roles that these FKBPs may play in

mediating γ-secretase component stabilities. Similarly, these experiments will confirm whether

inhibition of FKBPs in cell culture by FK506 affects components of the γ-secretase.

Another naturally derived immunosuppressant drug, known as Rapamycin, has been

shown to inhibit FKBP activites, similar to FK506 [223]. I suggest treating cells that express

components of the γ-secretase complex with Rapamycin, to inhibit FKBP functions. Analyzing

whether Rapamycin treatment will result in reduced protein levels of the γ-secretase

components, similar to that observed using FK506, will aid in our understanding of the roles of

FKBPs in γ-secretase protein stability.

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6.6 Conclusions

Genetic modifier screens have provided key players involved in Psn function, including

components of the Notch and APP signalling pathways. This has an impact on the study of

human diseases such as cancer and Alzheimer’s, since Notch and APP have been implicated in

these diseases. Elucidating the molecular mechanisms of FKBP functions may contribute to

therapies that help alleviate some of the symptoms of these disorders. Therefore, understanding

the cellular roles of individual proteins such as Psn and FKBP14 will provide crucial insights

into the mechanisms underlying human disease and development.

119

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the roles of presenilin and fkbp14 in drosophila ... · iv acknowledgements i would like to thank...

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