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Nuclear Localization of N-Ethylmaleimide Sensitive Factor by Marzena Serwin A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell & Systems Biology University of Toronto © Copyright by Marzena Serwin 2012

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Page 1: Nuclear Localization of N-Ethylmaleimide Sensitive Factor · Marzena Serwin Master of Science Cell & Systems Biology University of Toronto 2012 Abstract N-Ethylmaleimide Sensitive

       

Nuclear Localization of N-Ethylmaleimide Sensitive Factor

by

Marzena Serwin

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate

Department of Cell & Systems Biology University of Toronto  

© Copyright by Marzena Serwin 2012

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Nuclear Localization of N-Ethylmaleimide Sensitive Factor

Marzena Serwin

Master of Science

Cell & Systems Biology University of Toronto

2012

Abstract

N-Ethylmaleimide Sensitive Factor (NSF) was first characterized as the protein

responsible for restoring vesicle trafficking to N-ethylmaleimide (NEM) treated cells.

Diverse lines of evidence indicate that NSF may function in the nucleus of cells, a

previously unknown site of action for the protein. In this study Chinese hamster ovary

(CHO) cells were used to examine the cellular localization of NSF in fibronectin-spread

cells and in cells treated with NEM and leptomycin B (LMB). We show that NSF

localizes to the nuclei of several cell lines and cell types and shows variability in the

level of nuclear localization during cell spreading and drug treatments. Additionally, we

have identified three putative nuclear export sequences (NES) within the NSF-D2

domain. Furthermore, immunoprecipitation identified Vimentin as a likely nuclear

binding partner. Thus, for the first time, we report nuclear localization of NSF and an

uncharacterized interaction of NSF with Vimentin.  

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Acknowledgments

I would like to thank my supervisor, Professor Bryan Stewart, for his vast

reserve of knowledge and enduring guidance throughout my studies, and for his valuable

assistance during the writing of this thesis. His passion for science and his understanding

towards his students inspired me to pursue this degree and always work to improve upon

the project.

The members of my committee, Professor Rene Harrison and Professor Voula

Kanelis, have been instrumental in the completion of this work. Their insight and

reliability contributed to my success.

I would also like to express my deep gratitude to the past and present members

of the Stewart Lab: Dr. Xinping Qiu, Colin DeMill, Marta Kisiel and Dr. Sara

Seabrooke, to my brothers: Michal Serwin, Mateusz Serwin, Marcin Serwin OMI and

Marek Serwin and to Joshua Lopes for their constant guidance, support and friendship.

This work is a testament of their encouragement and never faltering aid.

Many thanks to Professor Mary Cheng, Professor George Espie, Professor

Angela Lange, Professor Joel Levine, Professor Tim Westwood and to their students,

who were always very kind and accommodating in allowing me to use their lab

equipment.

Finally, I would like to dedicate this thesis to my parents, without whom none of

this would be possible.

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Table of Contents

Abstract…………………………………………………………………………………..ii

Acknowledgments……………………………………………………………………….iii

Table of Contents………………………………………………………………………..iv

List of Figures…………………………………………………………………………...vi

List of Appendices……………………………………………………………………..viii

List of Abbreviations…………………………………………………………………….ix

Chapter 1 Nuclear Localization of N-Ethylmaleimide Sensitive Factor…………………1

1 Introduction…………………………………………………………………………….1

1.1 General……………………………………………………………………………..1

1.2 ATPases Associated with Diverse Cellular Activites (AAA/AAA+) Family of

Proteins………………………………………………………………………….....4

1.3 NSF and the SNARE Complex…………………………………………………….5

1.4 NSF: Structure and Function………………………………………………………5

1.5 Other Roles of NSF………………………………………………………………10

1.6 Nucleocytoplasmic Shuttling…………..…………………………………………12

1.7 Further Data from the Stewart Lab……………………………………………….13

1.8 Hypothesis………………………………………………………………………..17

2 Materials and Methods…………………………………………………………...…17

2.1 Culture Methods………………………………………………………………….17

2.2 Fibronectin Treatment……………………………………………………………18

2.3 Leptomycin B Treatment…………………………………………………………18

2.4 N-ethylmaleimide Treatment……………………………………………………..18

2.5 Cell Synchronization by Nocodazole Treatment…………………………………19

2.6 Cell Synchronization by Thymidine Treatment………………………………….19

2.7 Cellular Fractionation…………………………………………………………….19

2.8 Immunoprecipitation……………………………………………………………...21

2.9 Immunoblot Quantification………………………………………………………22

3 Results………………………………………………………………………………23

3.1 Optimization of Cellular Fractionation Protocol…………………………………23

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3.2 NSF Shows Nuclear Localazation and is affected by Fibronectin Mediated Cell

Spreading…………………………………………………………………………23

3.3 Bioinformatics Analysis of NSF………………………………………………….28

3.4 N-ethylmaleimide Affects Cellular Localization of NSF………………………...28

3.5 Leptomycin B Does Not Affect Nucleocytoplasmic Shuttling of NSF…………..31

3.6 NSF Shows Nuclear Localization Across Several Cell Lines……………………34

3.7 Nuclear NSF Interacts with Vimentin……………………………………………37

3.8 NSF is Present in the Nuclei of Prophase Cells…………………………………..40

4 Discussion…………………………………………………………………………..46

4.1 Overview…………………………………………………………………...……..46

4.2 Bioinformatics Analysis of NSF………………………………………...………..47

4.3 Fibronectin-Mediated Cell Spreading Affects NSF Localization……..…………47

4.4 NEM Affects the Subcellular Localization of NSF………………………………48

4.5 Exportin 1 is Likely Not Responsible for Nuclear-NSF Export………………….48

4.6 Evidence for Cytoplasmic Post-Translational Modification of NSF………..........49

4.7 NSF Localizes to the Nuclei of Various Cell Lines & Cell Types………….........49

4.8 Vimentin: A Putative Nuclear Binding Protein of NSF…………………….........50

4.9 NSF is Present in the Nuclei of Prophase Cells……………………………..........52

5 Summary and Conclusions………………………………………………………….54

References…………………………………………………………………………..56

Appendix……………………………………………………………………………71

                         

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List of Figures

Figure 1. Mapping of a NSF protomer…………………………………………………...3

Figure 2. Cartoon schematic showing stages of vesicle fusion………………………….6

Figure 3. Surface features of the NSF-D2 domain………………………………………7

Figure 4. Surface features of the NSF-N domain……………………………………..…8

Figure 5. Sequence alignment of the putative NSF-NES sequences (1, 2, and 3) of Homo

sapiens, Cricetulus griseus, Drosophila melanogastor, Dictyostelium discoideum

and Saccharomyces cerevisiae………..……………………………………………14

Figure 6. Sequence alignment of the putative NSF-NES1 of Drosophila melanogastor

and the NESs of α-actin, PKIα and MAPKK...……………………………………15

Figure 7. Imaging of mammalian-NSF nuclear localization within Drosophila Schneider

2 (S2) cells…............................................................................................………….16

Figure 8. A representative blot generated using the Bendeck Protocol.………………..24

Figure 9. A representative blot generated using the REAP Protocol…………………...25  

Figure 10. Transmitted light microscopy of non-spread and spread CHO cells……..…26

Figure 11. Whole cell, cytoplasmic and nuclear fractions from fibronectin spread or

not-spread CHO cells…….………………………………………………...………27

Figure 12. N-ethylmaleimide Sensitive Factor (residues 224-742) illustrating the three

nuclear export sequences and percentage of solvent accessible surface area……...29

Figure 13. N-ethylmaleimide Sensitive Factor D2 monomer illustrating the three nuclear

export sequences…………………………………………………………………...30

Figure 14. Whole cell, cytoplasmic and nuclear fractions from NEM treated or not-

treated CHO cells…………………………………………………………………..32

Figure 15. Imaging of wtNSF-EGFP in CHO cells in the absence, presence and post-

LMB……….……………………………………………………………………….33

Figure 16. Whole cell, cytoplasmic and nuclear fractions from LMB treated or not-

treated CHO cells…………………………………………………………..………35

Figure 17. Immunostaining of NSF within nuclear lysate originating from several cell

types………………………………………………………………….…………….36

Figure 18. N-Ethylmaleimide Sensitive Factor IP from CHO cells……...……..……...38

Figure 19. Vimentin IP from CHO cells.……...…………......…...…………………….39

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Figure 20. Nocodazole mediated CHO cell synchronization…………………………...42

Figure 21. Thymidine mediated CHO cell synchronization………………………........43

Figure 22. N-Ethylmaleimide Sensitive Factor IP from thymidine synchronized CHO

cells………………………………………………………………………………....45

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List of Appendices

Recipe of Necessary Buffers for Cellular Fractionation Protocol as per Lamond Lab (University of Dundee, Dundee, Scotland)……......………………………...………71

Permission for Reprint of Copyrighted Figures………………………………………...72

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List of Abbreviations

µl……………………………………………………...………………………..Microliters

%………………………………………………….………………………………..Percent oC…………………………………………………………………………Degrees Celsius

mg………………………………………………....…………………………...Milligrams

AAA……………………...………..ATPases Associated with Diverse Cellular Activities

BCA……………………...…………………………..………………..Bicinchoninic Acid

C………………………………...………………….……………….Cytoplasmic Fraction

CHO cells………………………………………...………...Chinese Hamster Ovary Cells

CRM1………………………………………..……..Chromosome Region Maintenance 1

DAPI……………………………………...…………..…...4',6-diamidino-2-phenylindole

DMEM………………………………...……………Dulbecco’s Modified Eagle Medium

DTT…………………………………..……………..………………………Dithiothreitol

FBS………………………………...………………..………………..Fetal Bovine Serum

FN……………………………..…………………..…………………………..Fibronectin

GFP…………………………………………………………….Green Fluorescent Protein

IP……………………………...……………………………………..Immunoprecipitation

Jurkat…………………………………………...Human T Cell lymphoblast-like cell line

kDa……………………………...……………………..……..………………..Kilodaltons

LMB…………………………..…………………………..………………..Leptomycin B

MAPKK………………..…….………………..Mitogen Activated Protein Kinase Kinase

N………………………………..………………..………..……………..Nuclear Fraction

N2A……………………………..………………..Cells Mouse Neuroblastoma N2A cells

NEM……………………………..……………….…..………………..N-Ethylmaleimide

NES……………………………...………...…..………………..Nuclear Export Sequence

NIH3T3……………………...…....………………..Mouse embryonic fibroblast cell line

NLS………………………………………………………Nuclear Localization Sequence

NPC………………………………………………………………..Nuclear Pore Complex

NSF…………………………..……..………………..N-Ethylmaleimide Sensitive Factor

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PC12…………………………..………………..Rat adrenal pheochromycytoma cell line

PKIα……………………………...…………..………………..Protein Kinase Inhibitor α

SNAP……………………………...…..………………..Soluble NSF Attachment Protein

SNARE…………………..…..…..…………..Solube NSF Attachment Protein Receptors

TBP…………………………………...………….………………..TATA Binding Protein

REAP………………………………………………………Rapid, Efficient and Practical

VAMP……………………….......………………..Vesicle Associated Membrane Protein

VCP…………………………..……………..………………..Valosin-Containing Protein

W……………………………...………………….....………………..Whole Cell Fraction  

       

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Chapter 1 Nuclear Localization of N-Ethylmaleimide Sensitive Factor

1 Introduction

1.1 General Within the cytoplasm of the eukaryotic cell reside organelles, each responsible

for a unique function. Transport between and within these structures requires membrane

fusion events. In the late 1970’s some key studies were conducted that addressed the

questions surrounding vesicle transport and specifically, membrane fusion events.

Siddiqi and Benzer (1976) first identified the Drosphila melanogaster comatose

temperature-sensitive mutant. The flies were exposed to a mutagen, ethyl

methanesulfonate, and the adult offspring of the mutagen-exposed parents were

incubated at nonpermissive temperatures (Siddiqi, O. and Benzer, S., 1976). This

resulted in conditional paralysis of some flies. However, when compared to other

temperature-sensitive mutants, the comatose mutant required a longer time to reach

paralysis and a longer time for recovery (Siddiqi, O. and Benzer, S., 1976). A few years

later Novick et al. (1980) identified 23 gene products that are involved in vesicle

secretion in the yeast Saccharomyces cerevisiae, among them Sec18-1. A decade later

Block et al. (1988) purified N-Ethylmaleimide Sensitive Factor (NSF) from Chinese

Hamster Ovary (CHO) cells, this protein would later be found to be responsible for the

comatose and Sec18-1 phenotype.

When Siddiqi and Benzer (1976) first characterized the D. melanogaster

comatose mutant they noted that the mutant slowly reached paralysis at restrictive

temperatures and slowly recovered when returned to permissive temperatures (Siddiqi,

O. and Benzer, S., 1976). Although, it was not known at the time D. melanogaster has

two NSF isoforms, unlike most organisms. Further characterization of the comatose

mutant identified the temperature-sensitive alleles to be within dNSF-1, a gene coding

for a NSF isoform (Pallanck, L. et al. 1995b). Despite there being an accumulation of

vesicles at the synapse of the comatose mutant, they cannot undergo exocytosis (Siddiqi,

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O. and Benzer, S., 1976). Similarly, mutagenesis studies conducted using S. cerevisiae

mutants showed an accumulation of vesicles within the cytoplasm, suggesting that the

23 genes targeted are involved in vesicle trafficking (Novick, P. et al. 1980). Among

them was Sec18-1, which gives rise to Sec18p, the yeast homolog of NSF (Novick, P. et

al. 1980). Rothman and colleagues (1989) decided to test whether vesicle trafficking

could be restored to the Golgi networks of NEM-treated CHO cells using cytosol

extracted from S. cerevisiae (Wilson, D.W. et al. 1989). The presence of the NSF yeast

homolog, Sec18p, restored vesicle transport (Wilson, D.W. et al. 1989). This confirmed

the functional equivalence of NSF and Sec18p, and suggested that similar vesicle

trafficking pathways exist in mammals and yeast (Wilson, D.W. et al. 1989).

Block et al. (1988) found that NSF was the catalytic protein capable of restoring

vesicular transport to Golgi networks treated with N-ethylmaleimide (NEM). Several

studies conducted over the next decade linked NSF to vesicle trafficking events from:

the endoplasmic reticulum to the Golgi apparatus, within the Golgi networks, in the

endocytic pathway, in neurotransmission and in neuroendocrine secretion (Beckers, C.J.

et al. I989; Malhotra, V. et al. 1988; Block, M. et al. 1988; lkonen, E. et al. I995; Diaz,

R. et al. I989; Pallanck, L. et al. 1995a; Moriyama, Y. et al. 1995). The data presented

by Block et al. (1988) helped characterize the mechanism by which NSF functions. The

study demonstrated that NSF was inhibited by the sulfhydryl alkylating agent, NEM,

suggesting a cysteine residue must be either important to NSF function or, at the very

least, near the functional site (Block, M.R. et al. 1988). Secondly, ATP-deprived

cytosolic fractions did not restore vesicular transport to NEM treated membranes,

suggesting that NSF requires ATP for its functioning (Block, M.R. et al. 1988).

NSF is a 78kDa ATPase Associated with diverse cellular Activities (AAA)

protein with a homoheaxmeric structure. NSF contains three domains (NSF-N, NSF-D1

and NSF-D2) (Figure 1). NSF-N (residues 1-205) was identified as the site of α-soluble

NSF attachment proteins (α-SNAPs) - Soluble NSF Attachment Protein Receptors

(SNARE) binding (May, A.P. et al. 1999). NSF-D1 (residues 206-488) was found to be

the most active ATPase domain of NSF, while NSF-D2 (residues 489-744) was found to

be necessary for NSF hexamerization, the latter two identified NSF as a member of the

AAA family (Tagaya, M. et al. 1993).

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Figure 1. Domain Map of an NSF protomer. Shown are the N-terminal, D1 and D2 domains and the Walker B and Walker A motifs within the D1 and D2 domains, respectively. Putative NESs within the D2-domain are amplified.

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1.2 ATPases Associated with Diverse Cellular Activities (AAA and AAA+) Family of Proteins

The proteins composing the AAA family are characterized by a 200-250 conserved

amino acid ATP binding domain, the AAA domain (Hanson, P.I. and Whiteheart, S.W.

2005). Within this sequence lie the Walker A and Walker B motifs, which facilitate the

ATPase activity of the AAA family members (Hanson, P.I. and Whiteheart, S.W. 2005).

Further structural characterization of the classic AAA proteins has led to the

establishment of a more diverse superfamily, now identified as the AAA+ family

(Hanson, P.I. and Whiteheart, S.W. 2005). Present within all kingdoms, the AAA+

ATPases are oligomers and often undergo hexamerization (Lenzen, C.U. et al. 1998;

Hanson, P.I. and Whiteheart, S.W. 2005).

There are some motifs that are present within the subfamily of AAA ATPases,

but are not common to all AAA+ proteins. One example of a classic AAA motif is the

second region of homology (SRH), positioned toward the C-terminal of the Walker B

motif (Lupas, A.N. and Martin, J. 2002). NSF contains the SRH motif and so falls

within the classification of the AAA family of proteins (Lupas, A.N. and Martin, J.

2002). However, common to all AAA+ proteins is the Sensor 1 motif positioned toward

the C-terminal of the Walker B motif (Steel, G.J. et al. 2000). Additionally, the Walker

A and B motifs are crucial for the ATPase activity of the AAA+ proteins (Neuwald, A.F.

et al. 1999).

The P-loop, contained within the Walker A motif, contains a consensus sequence

GXXXXGK[T/S], where X represents any amino acid. Point mutations of the lysine to

an alanine (K to A) abolishes ATP binding within the Walker A motif (Babst, M. et al.

1998). Similarly, within the Walker B motif lies the ATP coordinating sequence

hhhhDE (where h represents a hydrophobic residue). The aspartate coordinates the

magnesium ion, while glutamate activates the water molecule necessary for the

hydrolysis reaction. Point mutating glutamate at position 326 to glutamine (E326Q)

results in an AAA+ protein that is capable of binding ATP, but not catalyzing its

hydrolysis (Weibezahn, J. et al. 2003). The E/Q mutation is commonly used in the study

of NSF function (Dalal, S. et al. 2004; Stewart, B.A. et al. 2001).

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1.3 NSF and the SNARE Complex

Discovery of NSF ushered in rapid identification of proteins involved in vesicle

trafficking and membrane fusion (Clary, D.O. et al. 1990; Söllner, T. et al. 1993a;

Söllner, T. et al. 1993b). Among these discoveries, the Soluble NSF Attachment Protein

(SNAPs) and the SNAP receptors (SNAREs) proved critical for development of the

SNARE hypothesis (Söllner, T. et al. 1993b; Rothman, J.E. and Warren, G. 1994). The

SNAREs, thought to be the key mediators of membrane fusion, are grouped into vesicle

membrane associated SNARE (v-SNARE) and target membrane associated SNAREs (t-

SNAREs). It is the association of Synaptobrevin, also known a vesicle associated

membrane protein (VAMP), a v-SNARE, with Syntaxin and Synaptosomal Associated

Protein 25 (SNAP-25), both t-SNAREs, that drives membrane fusion, leading to

exocytosis. Upon fusion the v-SNARE and t-SNAREs reside as a protein complex in the

same membrane; this complex is known as the cis-SNARE complex. In order for these

proteins to participate in further rounds of exocytosis they must be unraveled from one

another. NSF accomplishes this task via three adaptor protein α-SNAP per NSF

hexamer, this has earned NSF the title of a chaperone protein, a protein that assists in the

disassembly of protein complexes (Moeller, A. et al. 2012) (Figure 2).

1.4 NSF: Structure and Function

Crystal structures of the NSF-D2 (1.75 Å) and NSF-N (1.9 Å) domains have been

determined and 11 Å electron cryomicroscopy and single-particle analysis data exist for

the NSF-hexamer and the 20S particle (Yu, R.C. et al. 1998; Lenzen, C. et al. 1998;

May, A.P. et al. 1999; Yu, R.C. et al. 1999; Hanson, P.I. et al. 1997; Hohl, T.M. et al.

1998; Furst, J. et al. 2003) (Figures 3 and 4). The NSF-hexamer is composed of three-

layers, (from the N-terminal to the C-terminal) the N-domains, D1-domains and D2-

domains with an approximate diameter of 115Å, though the D1- and D2-domains show

changes in diameter between the ATP and ADP-bound states (Moeller, A. et al. 2012;

Chang, L.-F. et al. 2012). The D1- and D2-domains have a parallel arrangement with

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Figure 2. Cartoon schematic showing stages of vesicle fusion. Vesicle docking and priming, membrane fusion followed by NSF-mediated SNARE complex dissociation and recycling is shown.

!

!

Docking Priming Fusion

NSF-mediated SNARE Disassembly

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Figure 3. Surface features of the NSF-D2 domain. (A) N-terminal face of NSF-D2. (B) C-terminal face of NSF-D2. The crystal structure was resolved at 1.75 Å. Reprinted with permission (Lenzen, C. U., et al. 1998).

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Figure 4. Surface features of the NSF-N domain. (B) Sequence conservation of exposed NSF-N surface. (C) Identified grooves on NSF-N surface, with groove 3 being a likely binding site of α-SNAP. The crystal structure was resolved at 1.9 Å. Reprinted with permission (Yu, R. C. et al. 1999).

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respect to each other, much like the p97 D1- and D2-domains (Moeller, A. et al. 2012;

Chang, L.-F. et al. 2012). The 20S complex resembles a sparkplug, with the three α-

SNAPs forming a tripod-like structure that binds the SNARE complex at its C-terminus,

while its N-terminus is anchored by the N-domain (Moeller, A. et al. 2012; Chang, L.-F.

et al. 2012).

Though all of the details of NSF mediated SNARE disassembly are not clear, a

recent study by Whiteheart and colleagues brought to light a possible mechanism

(Moeller, A. et al. 2012). Structural modeling, using electron microscopy analysis, of the

20S particle in the ATP-bound pre-SNARE-disassembly state (using a non-hydrolyzable

ATP-analog, AMP-PNP) and in the ADP-bound post-SNARE disassembly state,

showed conformational changes occurring in the N- and D1-domains, with the D2-

domain showing no change (Moeller, A. et al. 2012). Specifically upon ATP-hydrolysis,

via the D1-domain, the N-domain residues identified as α-SNAP-binding sites move

from an upward exposed position to a downward inaccessible position (Moeller, A. et al.

2012). This suggests that SNARE complex disassembly is strongly dependent on the

nucleotide state of NSF-D1; an ATP-bound state facilitates SNAP-SNARE binding,

while an ADP-bound state prevents it (Moeller, A. et al. 2012; Chang, L.-F. et al. 2012).

Similarly to the Whiteheart group, Chang et al. (2012) also used structural

analysis of NSF in various nucleotide states to analyze the mechanics of NSF-mediated

SNARE complex disassembly. Using wild-type NSF from CHO cells the authors

reconstructed the protein from unbound state NSF monomers and confirmed the NSF

proteins were functional and formed hexamers in homogeneous ATPγS (a

nonhydrolyzable ATP-analog), ADP-AlFx (a transition state ATP-analog) and ADP-

bound states (Chang, L.-F. et al. 2012). Chang et al. (2012) identified an exposed,

SNAP-SNARE competent upward binding state, and inaccessible downward state of the

NSF-N domains, much like the changes seen by Moeller, A. et al. (2012).

Additionally, both groups confirmed a D1-domain rotation occurring during

ATP-hydrolysis, suggesting this is the mechanical force yielded from ATP hydrolysis

and required to pry the SNARE complex into its components (Moeller, A. et al. 2012;

Chang, L.-F. et al. 2012). The D1-N-linker shows a high degree of flexibility and likely

serves to transfer the mechanical force derived from ATP-hydrolysis from the D1-

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domain to the N-domain, which eventually pulls the SNARE complex apart resulting in

the downward conformation; this motion has been referred to as the ‘power stroke’

(Moeller, A. et al. 2012; Chang, L.-F. et al. 2012). Three-dimensional reconstructions of

the D2-domain using electron microscopy imaging showed no changes in all three

nucleotide states tested, suggestive of the rigidity of this domain (Moeller, A. et al.

2012; Chang, L.-F. et al. 2012). In contrast, significant conformational changes were

observed in the D1- and N-domains (Moeller, A. et al. 2012; Chang, L.-F. et al. 2012).

Therefore, the authors of both studies suggest that the D2-domain likely serves as a

platform on which the disassembly occurs (Moeller, A. et al. 2012; Chang, L.-F. et al.

2012).

Although, much information has been accumulated regarding NSF’s structure

and function there remain unaddressed results that leave inconsistencies in the NSF

story. Several studies have reported NSF’s relocalization from the soluble to insoluble

fraction (Block, M. et al. 1988; Mohtashami, M. et al. 2001; Liu, C. and Hu, B. 2004).

N-Ethylmaleimide Sensitive Factor relocalization has been linked to the inactivation of

NSF however no further studies have been conducted on insoluble NSF. Insoluble NSF

may in fact be NSF that is associated with the nuclear membrane, as it was shown by

Tagaya et al. (1996), rather than simply a dysfunctional NSF form (Tagaya, M. et al.

1996; Mashima, J. et al. 2000). Other possibilities to consider are that NSF may

association with other proteins leading to the formation of insoluble complexes.

Furthermore, no studies to date have examined the possibility of NSF nuclear

localization, although data suggest this is feasible. These results and hypotheses require

additional investigation to elucidate further NSF’s cellular roles.

1.5 Other Roles of NSF

In addition to its SNARE-dependent role, NSF also interacts with non-SNARE proteins

and shares sequence similarity with other AAA proteins, suggestive of other roles it may

play within the cell (Whiteheart, S.W. et al. 2004). In particular, the N-domains of p97

and valosin-containing protein (VCP) are highly conserved to that of NSF and all three

play important roles in membrane fusion events in the cytoplasm (Whiteheart, S.W. et

al. 2004; Partridge, J.J et al. 2003). Furthermore, p97/VCP has been found to act as a

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chaperone for ubiquitinated proteins destined for proteasomal degradation following

ubiquitination by E3 ubiquitin ligase gp78 (Fang, S. et al. 2001; Zhong, X. et al. 2004).

In 2004, Zhong et al. furthered our understanding of this interaction by showing

that gp78 and p97/VCP physically interact (Zhong, X. et al. 2004). NSF has also been

shown to interact with an E3 ubiquitin ligase, Highwire, responsible for regulation of

Wallenda levels, a protein that plays a major role in synaptic growth (Collins, C. et al.

2006; Kaneuchi, T. et al. unpublished data). Loss-of-function Highwire mutants show

synaptic overgrowth at the Drosophila melanogaster neuromuscular junction (Collins,

C. et al. 2006). This phenotype is also shown in the NSFE/Q mutant, which is capable of

binding, but not hydrolyzing ATP (Kaneuchi, T. et al. unpublished data). Work done by

Kaneuchi, T. et al. suggests that NSF acts in the same pathway as Highwire in the

suppression of the synaptic overgrowth phenotype. The authors of this study postulate

that NSF’s role in this pathway may involve the disassembly of Highwire from its

substrates (Kaneuchi, T. et al. unpublished data). Additionally, Lowenstein and

colleagues (2003) have demonstrated that S-nitrosylation of NSF inhibits secretion of

Weibel-Palade bodies from endothelial cells (Matsushita K. et al. 2003). N-

Ethylmaleimide Sensitive Factor has also been shown to mediate rapid surface

expression of AMPA subunit, GluR2, thereby preventing long-term depression (Kamboj

S.I. et al. 1998; Nishimune A. et al. 1998; Osten P. et al. 1998; Huang Y. et al. 2005).

Though many AAA+ proteins have been found to localize to the nucleus, AAA

proteins were not known for their nuclear roles. In 2003, Indig and colleagues reported

that p97/VCP localizes and is abundant in the mammalian nuclei, functioning as a

regulator of nucleic acid recycling (Partridge, J.J. et al. 2003). In 1996 and 2002 Tagaya

and colleagues reported the presence of NSF in the nuclear membrane of PC12 cells

(Tagaya, M. et al. 1996; Mashima, J. et al. 2000). Additionally, these results were

confirmed when isolated nuclei from bovine adrenal medulla showed NSF presence in

the nuclear membrane (M. Tagaya and S. Mizushima, unpublished data). Therefore,

although there are few reports on the role of nuclear NSF, the strong similarity and

currently known cellular functions of NSF and p97/VCP suggest that localization of

NSF to the nucleus is feasible. Moreover, it is highly unlikely that AAA proteins, which

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show congruence in structure and function to NSF should play several cellular roles,

while NSF is limited to SNARE disassembly.

1.6 Nucleocytoplasmic Shuttling

The nucleoplasm and cytoplasm are separated by the bilayered nuclear envelope

(Kumeta, M. et al. 2012). Nuclear export and import occur through the nuclear pore

complex (NPC), a 40-50nm in diameter channel that serves to exclude some material,

while facilitating the transport of other (Güttler, T. and Görlich, D. 2011). The octameric

NPC is composed of approximately 30 different proteins known as nucleoporins (Nups)

(Xu, L. and Massagué, J. 2004; Kumeta, M. et al. 2012).

Nuclear import involves the binding of importins to the nuclear localizing

sequence (NLS) of their cargo (Rexach, M. and Blobel, G. 1995; Görlich, D. et al.

1996). Once the dimer is formed it travels from the cytoplasm through the NPC into the

nucleoplasm. There, the high concentration of RanGTP facilitates the displacement of

the cargo with RanGTP (Rexach, M. and Blobel, G. 1995; Görlich, D. et al. 1996). Ran

(or RAs-related Nuclear protein) is a 25kDa protein involved in nucleocytoplasmic

transport (Kutay, U. et al. 1997; Floer, M. et. al. 1997). Ran exists in two states,

RanGTP and RanGDP. The nucleus has a RanGTP concentration 1000-fold higher than

that seen within the cytoplasm (Kutay, U. et al. 1997; Floer, M. et. al. 1997). Once the

importin is RanGTP bound, it travels back to the cytoplasm via the NPC and the GTPase

activity is stimulated allowing the dimer to dissociate (Rexach, M. and Blobel, G. 1995;

Görlich, D. et al. 1996).

Nuclear export of larger molecules requires that the cargo contain a NES. The

pathway that cargo travels when moving from the nucleoplasm into the cytoplasm

depends on GTP-bound Ran (Kutay, U. et al. 1997; Floer, M. et. al. 1997). The high

nuclear RanGTP concentration acts to stimulate exportin-cargo binding, similarly, cargo

binding stimulates RanGTP binding (Kutay, U. et al. 1997; Floer, M. et. al. 1997;

Güttler, T. and Görlich, D. 2011). This complex travels through the NPC. Once in the

cytoplasm the intrinsic GTPase activity of Ran is stimulated by the binding of

RanGTPase-activating protein (RanGAP) (Kutay, U. et al. 1997; Floer, M. et. al. 1997).

However, RanGAP can itself only interact with exportin-bound RanGTP by binding to

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Ran-binding proteins, also known as Ran-binding domains (RanBDs) (Kutay, U. et al.

1997; Floer, M. et. al. 1997). This results in the hydrolysis of GTP to GDP and

disassembly of the export complex.

The most well studied exportin is CRM1, commonly referred to as Exportin 1

(Fukuda, M. et al. 1997; Stade, K. et al. 1997). Unlike other exportins that have a

narrow cargo list, Exportin 1 caters to a wide variety of unrelated cargo (Fukuda, M. et

al. 1997; Stade, K. et al. 1997). Hydrophobic residues with leucine/isoleucine residues

at distinct positions characterize the NES recognized by Exportin 1 (Fukuda, M. et al.

1997; Stade, K. et al. 1997).

Our analysis revealed that NSF has three putative, previously unknown, nuclear

export sequences (NESs) within the NSF-D2 domain (Figure 1; Xinping, Qiu,

unpublished data) and BLAST sequence alignment of the putative NSF-NES sequences

within five species was conducted (Figure 5). Conserved hydrophobic residues were

identified at the 2nd, 6th and 9th position between all three NESs and across all species

considered, as seen in the established NESs of Exportin 1 cargo: α-actin (NES-1/NES-

2), PKIα and MAPKK (Wada, A. et al., 1998; Figure 6). When point mutations within

the mammalian NSF-D2 domain of nuclear export sequence 3 were introduced, there

was an increase of NSF nuclear signal within Drosophila Schneider 2 (S2) cells, when

compared to wtNSF (Figure 7; Xinping, Qiu, unpublished data).

1.7 Further Data from the Stewart Lab

Genetic screens in our lab using a Gene Search collection of a Drosophila

melanogaster-specific transposable element, P-element, insertion followed by more

specific genetic crosses, have shown that NSF interacts with nuclear proteins (some

examples include transcription factors: E2f, ovo, btd, btn, lola, dpld, fos, jun and other

such as DNA binding proteins such as: LanA and His2Av) (Laviolette, M.J. et al. 2005;

Peyre, J.B. et al. 2006). Moreover, unpublished work on NSF-induced morphology

phenotypes showed, that over-expression of Fos and Jun transcription factors suppressed

overgrowth at the larval D. melanogaster neuromuscular junction (Qiu et al.

unpublished). Altogether, several lines of indirect evidence suggest NSF may play a role

in the nuclear function of the cell.

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Figure 5. Sequence alignment of the putative NSF-NES sequences (1, 2, and 3) of Homo sapiens, Cricetulus griseus, Drosophila melanogastor, Dictyostelium discoideum and Saccharomyces cerevisiae. Conserved hydrophobic residues are boxed; residues that are not conserved are in red. Sequence alignment was conducted using BLAST.

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Figure 6. Sequence alignment of the putative NSF-NES1 of Drosophila melanogastor and the NESs of α-actin, PKIα and MAPKK. Conserved hydrophobic residues are boxed. The NESs of α-actin (1 and 2), PKIα and MAPKK were reported in Wada, A. et al., 1998.

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Figure 7. Imaging of mammalian-NSF nuclear localization within Drosophila Schneider 2 (S2) cells. (A) Nuclear envelope staining using Lamin Dm0 (B) Mammalian wtNSF-EGFP (top) and mammalian NSF-EGFP with a point mutation within the NES3 sequence (I705A and I707A) (bottom) (C) Overlay of A and B (Xinping Qiu, unpublished data).

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In order to more explicitly show nuclear localization of NSF and further

delineate NSF function, we turned to cell culture studies. In one series of experiments

that examined NSF in a cell-spreading model, we detected NSF in the nuclei of cells

only after spreading occurred. This suggested that NSF has a dynamic, previously

undetected localization in cells.

1.8 Hypothesis

In 1996 NSF was first shown to localize to the nuclear membrane of PC12 cells and

recently, we have identified three putative nuclear export sequences (NESs) within the

NSF-D2 domain (Figure 1; Xinping, Qiu, unpublished data). Continuing with this body

of work, from both cell culture and D. melanogaster studies, my working hypothesis is

that NSF is a dynamic nuclear protein that interacts with other nuclear proteins to

control cell function. To test this hypothesis I used a cell-fractionation approach to

examine nuclear localization in spread versus non-spread cells of NSF and in cells

treated with drugs known to inhibit nuclear export. Secondly, to identify potential NSF

interacting proteins, I immunoprecipitated NSF and used mass spectrometry to identify

its nuclear-specific binding partners. Thirdly, I used nuclear fractions originating from

several cell lines, in order to determine if NSF is present in the nuclei of a wide variety

of cell types. Lastly, I tested the physiological importance of nuclear NSF by examining

NSF’s interacting partners during specific phases of the cell cycle.

My results show that NSF localizes to the nuclei of Chinese hamster ovary

(CHO-K1) cells, neuroblastoma N2A cells, PC12 (rat adrenal pheochromycytoma) cells

and NIH 3T3 (mouse embryonic fibroblast) cells. Using immunoprecipitation, I have

identified Vimentin as a nuclear binding partner of NSF.

2 Materials and Methods

2.1 Culture Methods

Chinese hamster ovary (CHO) cells and mouse neuroblastoma N2A (N2A) cells were

grown at 36.5oC, 5% CO2 and 65% humidity. CHO cells were grown in Dulbecco’s

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Modified Eagle Medium (DMEM) (Sigma, Cat#D5546) containing 10% Fetal Bovine

Serum (FBS) (Sigma, Cat#F6178). N2A cells were grown in high glucose DMEM

(Invitrogen, Cat#10567-014) containing 10% FBS and 1% penicillin-streptomycin

(Sigma, Cat#P0781). When cells reached 90% confluency the growth medium was

removed and cells were trypsinized (Sigma, Cat#T4049). The trypsin was removed and

6mL of fresh growth medium was added to the flask. Immediately after, 1mL was drawn

and a fresh flask containing 5mL of growth medium was inoculated to a maximum of 6

new culture flasks.

Stock cultures of CHO and N2A cells were harvested and stored at -80oC and

when necessary, thawed and cultured. CHO cells were stored at -80oC in 40% DMEM,

50% FBS and 10% Dimethyl sulfoxide (DMSO) (Bioshop, Cat#DM5555). N2A cells

were stored at -80oC in 40% high glucose DMEM, 50% FBS and 10% DMSO.

2.2 Fibronectin Treatment

CHO or N2A stock culture cells were split and two flasks were seeded. Cells were

grown as described in Culture Methods until approximately 90% confluency. Cells were

trypsinized and re-plated to a fibronectin-coated test plate or not-coated test plate and

incubated for 3 hours at 36.5oC, 5% CO2 and 65% humidity. Cellular fractionation

followed as described in Cellular Fractionation.

2.3 Leptomycin B Treatment

CHO stock culture cells were split and two flasks were seeded. Cells were grown as

described in Culture Methods until approximately 90% confluency. The growth medium

was removed and 4mL of fresh medium was added to each flask. Leptomycin B

(3.4x10-3 nM; Sigma, Cat#L2913) was added to one flask and the second remained as a

control. Cells were incubated at 36.5oC, 5% CO2 and 65% humidity for 4 hours. Cellular

fractionation followed as described in Cellular Fractionation.

2.4 N-ethylmaleimide Treatment

CHO stock culture cells were split and two flasks were seeded. Cells were grown as

described in Culture Methods until approximately 90% confluency. The growth medium

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was removed and 5mL of ice-cold 1xPBS pH 7.4 was added to each flask. NEM

(40µM/1mM/5mM; Sigma, Cat#E3876) was added to three experimental flasks and the

fourth remained as a control. Cells were placed on a rocker on ice for 15 minutes

followed by DTT (20mM/0.5M/25M; Sigma, Cat#D9779) quenching for 15 minutes on

a rocker on ice. Cellular fractionation followed as described in Cellular Fractionation.

2.5 Cell Synchronization by Nocodazole Treatment

CHO stock culture cells were split and three flasks were seeded. Cells were grown as

described in Culture Methods until approximately 80% confluency. Nocodazole

(50ng/ml; Sigma, Cat#M1404) was added directly to the growth medium. Cells were

incubated at 36.5oC, 5% CO2 and 65% humidity for 10 hours. Cellular fractionation

followed as described in Cellular Fractionation.

2.6 Cell Synchronization by Thymidine Treatment

CHO stock culture cells were split and four flasks and 19 test plates were seeded. Cells

were grown as described in Culture Methods until approximately 50% confluency.

Thymidine (2mM; Sigma, Cat#T1895) was added directly to the growth medium. Cells

were incubated at 36.5oC, 5% CO2 and 65% humidity for 24 hours. After the 24-hour

incubation, the thymidine-containing medium was removed from the culture flasks and

test plates and the cells were rinsed twice with growth medium. New growth medium

was added to each rinsed flask and test plate. The cells progression through the cell

cycle was monitored at each hour, with time 0 (T0) marking the removal of thymidine,

for a total of 18 hours. Cells grown on test plates were fixed with 4% formaldehyde and

stained with 4',6-diamidino-2-phenylindole (DAPI) The cells were incubated at 36.5oC,

5% CO2 and 65% humidity until they reached prophase. Cellular fractionation followed,

for the cells grown in flasks, as described in Cellular Fractionation.

2.7 Cellular Fractionation

Cellular fractionation was conducted as described earlier in Lamond Lab: Cellular

Fractionation, (http://www.lamondlab.com/pdf/CellFractionation.pdf,  Lamond Lab,

University of Dundee, Dundee, Scotland) with some modifications. In brief, DMEM

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was removed and cells were washed with ice-cold 1XPBS pH 7.4, and the solution was

gently removed. Ice-cold Buffer A (1mL) was added and cells were scraped off the

plates, transferred to an Eppendorf tube, kept on ice for 5 minutes and mechanically

disrupted using a Dounce homogenizer to create a whole cell lysate (Please refer to

Appendix A for Buffer A recipe). The whole cell lysate samples were aliquoted

(300µL), 60µL of 5X RIPA buffer was added to the samples, sonicated for 10 seconds

on ice (Duty Cycle: 20%; Timer: 2; Output Control: 3) (Branson Sonifier 250, VWR

Scientific), followed by centrifugation at 2800 g for 10 minutes at room temperature

using an Eppendorf table top Micromax centrifuge. A whole cell lysate sample was then

transferred to a fresh Eppendorf tube and stored at -80oC.

The remaining whole cell lysate sample was centrifuged at 228 g for 5 minutes at

room temperature and the supernatant was collected as the cytoplasmic fraction

(300µL). 60µL of 5X RIPA buffer was added to the cytoplasmic fraction and the sample

was centrifuged at 2800 g for 10 minutes at room temperature. The cytoplasmic sample

was then transferred to a fresh Eppendorf tube and stored at -80oC.

The remaining pellet was resuspended in S1 (600µL) solution and S3 (600µL)

solution was gently layered on top (Please refer to Appendix A for S1 and S3 recipe).

The sample was centrifuged at 2800 g for 10 minutes at room temperature. The

supernatant was collected and stored at -80oC, while the resulting nuclear pellet was

resuspended in 1X RIPA buffer (150µL) and then sonicated for 10 seconds on ice (Duty

Cycle: 20%; Timer: 2; Output Control: 3) (Branson Sonifier 250, VWR Scientific)

followed by centrifugation at 2800 g for 10 minutes at room temperature. This nuclear

sample was then transferred to a fresh Eppendorf tube and all samples (whole cell,

cytoplasmic, and nuclear fractions) were stored at -80oC.

Nuclear lysates originating from the human epithelial carcinoma cell line (HeLa;

Abcam, Cat#ab14655) and rat adrenal pheochromocytoma cell line (PC12; Abcam,

Cat#ab14884), mouse embryonic fibroblast cell line (NIH 3T3; Abcam Cat#ab14874),

and human T cell lymphoblast-like cell line (Jurkat; abcam, Cat#ab14844) were

purchased and resolved on a 10% SDS-PAGE gel followed by immunoblotting. Nuclear

lysates originating from CHO cells and N2A cells (N2A cells were a gift from Dr. Mary

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Cheng’s Lab at the University of Toronto Mississauga) were generated using the

Cellular Fraction protocol.

2.8 Immunoprecipitation

Protein G resin (50µL; Pierce, Cat#20398) was transferred to an Eppendorf tube,

washed with cold lysis buffer (450µL) and centrifuged at 10,000 g for 30 seconds. The

supernatant was discarded and the resin was resuspended in 50µL of cold lysis buffer

with complete, EDTA-free protease inhibitors (Roche Applied Science,

Cat#05056489001) and the nuclear or cytoplasmic fraction (500µL) was added and the

sample(s) were rocked on ice for 1 hour. After the 1 hour incubation the sample(s) was

centrifuged at 10,000 g for 2 minutes and the supernatant was divided equally between

two Eppendorf tubes containing newly washed protein G resin suspended in 50µL of

cold lysis buffer with complete, EDTA-free protease inhibitors. Special care was taken

so as to not disturb the pelleted protein G resin. Anti-NSF or anti-Vimentin (3µL;

Sigma, Cat#V6630) was added to one sample. A second sample was used as a negative

control and no antibody was added. The samples were incubated with rocking for 12-24

hours at 4oC.

After the incubation period the samples were centrifuged at 10,000 g for 1

minute and the supernatant was removed and kept as the flow through sample. The

beads were resuspended in 500µL of lysis buffer with complete, EDTA-free protease

inhibitors (Roche Applied Science, Cat#05056489001) and subsequently centrifuged at

10,000 g for 1 minute. The supernatant was removed and kept as wash 1; this was

repeated two more times and each time the supernatant was removed and kept as wash 2

and wash 3. After the last wash the protein G resin pellet was resuspended in 50µL of 1x

protein loading buffer and the sample was vortexed. Once the whole cell lysate fraction

(used as a positive control), flow through, wash 1, wash 2 and wash 3 samples were

aliquoted, 2x protein loading buffer was added to each sample and all samples were

boiled at 100oC for 10 minutes. The samples were then centrifuged at 10,000 g for 20

seconds and resolved by performing an SDS-PAGE (10% gel) followed by

immunoblotting. The blots were incubated with primary antibodies, anti-NSF and anti-

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Vimentin. Blots were subsequently washed and incubated with appropriate secondary

antibodies and signals revealed with ECL plus reagents.

2.9 Immunoblot Quantification

Protein concentrations were determined using a Bicinchoninic Acid (BCA) Protein

Assay kit (Pierce, Cat#23227) and their absorbencies measured with a nanodrop

spectrophotometer (Nanodrop Technologies, NanoDrop ND-1000 Spectrophotometer).

Samples of equal protein concentration, determined using BCA assay were resolved by

performing an SDS-PAGE (10% gel) followed by immunoblotting. The blots were

incubated with the appropriate primary antibodies: anti-N-Ethylmaleimide Sensitive

Factor (anti-NSF; Cell Signaling Technology, 1:5000, Cat#2145), anti-Vimentin (3µL;

Sigma, Cat#V6630), anti-Tubulin (E7 anti-Tubulin; dilution factor 1:200,

Developmental Studies Hybridoma Bank), anti-Actin (mAb JLA-20, 1:200,

Developmental Studies Hybridoma Bank) and anti-TATA Binding Protein (anti-TATA

BP; dilution factor 1:1000, Fisher-Scientific Cat#MA125427). Blots were subsequently

washed and incubated with appropriate secondary antibodies and signals revealed with

ECL plus reagents (GE Healthcare).

Separate immunoblotting of the whole cell, cytoplasmic and nuclear fractions

facilitated the use of ImageJ in order to measure NSF levels across the subcellular

fractions. Whole cell, cytoplasmic and nuclear NSF levels were quantified and any

treated fraction was divided by the corresponding control, yielding a quotient. When the

quotient value was 1, NSF levels remained unchanged between the treated and control

cells, when the quotient value was >1 there was an increase in NSF levels during the

treatment, and if the quotient value was <1 there was a decrease in NSF levels during the

treatment. It should be noted that the purification steps involved in the separation of the

cytoplasmic and nuclear fractions require several transfers of the sample. This reduces

contamination of the fractions, but results in a loss of sample. Consequently, changes in

the whole cell lysate best reflect the overall changes, however they do not necessarily

correspond to the changes seen in the cytoplasmic and nuclear fractions.

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3 Results

3.1 Optimization of Cellular Fractionation Protocol

The first objective of my research was to establish localization of NSF into the nucleus

of CHO cells using western blotting techniques. In order to determine this, I performed

cellular fractionation. Several protocols were tested: Abcam: Nuclear Fractionation

Protocol; Bendeck Lab: Nuclear Fractionation; REAP: A two minute cell fractionation

method and Lamond Lab: Cellular Fractionation (Abcam:

http://www.abcam.com/ps/pdf/protocols/Nuclear%20fractionation%20protocol.pdf;

Chow, W., et al. 2008; Suzuki, K. et al., 2010; Lamond Lab, University of Dundee,

2007). It took several trials to establish the protocol (Figure 8, 9 & 11A). The Bendeck

Lab: Nuclear Fractionation protocol produced poor isolation of the nuclear fraction as

evidenced from the lack of TBP staining in the nuclear fraction (Figure 8). Therefore, it

was necessary to use an alternate protocol. The REAP protocol, resulted in strong NSF

bands, however there was poor separation of the nuclear and cytoplasmic fraction as

TBP appears in all three fractions (Suzuki, K. et al., 2010; Figure 9). Consequently, the

Lamond Lab: Cellular Fractionation protocol was considered (Figure 11A).

In the Lamond Lab protocol no detergent was used until after fraction separation.

The hypotonic buffer used prior to dounce homogenization of cells, facilitated cell lysis

without dissolving the nuclear membrane, as a detergent buffer would do. The cellular

fractionation protocol provided by the Lamond Lab produced the most reliable results

and after optimizing this protocol for my needs and introducing some modifications, I

produced replicable results.

3.2 NSF Shows Nuclear Localization and is Affected by Fibronectin Mediated Cell Spreading

The purpose of this work was to investigate the nuclear localization of NSF and to

identify its binding partner(s) in efforts to characterize its nuclear role. Our confocal

imaging data had previously shown an increase in the nuclear accumulation of NSF in

fibronectin spread cells. Additionally, a study published by Skalski et al. (2005)

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Figure 8. A representative blot generated using the Bendeck Protocol (Chow et al., 2008). Whole cell (W), cytoplasmic (C) and nuclear (N) fractions from fibronectin (FN) spread or not-spread CHO cells. Cells were lifted and plated on FN coated or not-coated plates and incubated for 3 hours prior to fractionation. Fractionation was conducted using two different detergents in efforts to assess which one performed better; both showed comparable results. SDS-PAGE was performed followed by a western blot. White arrows indicate molecular weights in kDa.

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Figure 9. A representative blot generated using the REAP Protocol (Suzuki, K. et al., 2010). Whole cell (W), cytoplasmic (C) and nuclear (N) fractions from fibronectin (FN) spread or not-spread CHO cells. Cells were lifted and plated on FN coated or not-coated plates and incubated for 3 hours prior to fractionation. SDS-PAGE was performed followed by a western blot. White arrows indicate molecular weights in kDa.

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Figure 10. Transmitted light microscopy (Eclipse Nikon; lens Nikon Fluor 60x/1.00W) of non-spread (A) and spread (B) CHO cells. Cells were trypsinized and transferred to a non-coated test plate (A) or to a test plate coated with immobilized fibronectin (B); 2 hours later plates was imaged. Bars represent 20µm.

A B

!!!!! !

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Figure 11. Whole cell (W), cytoplasmic (C) and nuclear (N) fractions from fibronectin (FN) spread or not-spread CHO cells. Cells were lifted and plated on fibronectin coated or not-coated plates and incubated for 3 hours prior to fractionation. SDS-PAGE was performed followed by a western blot (A) 16µg of protein was loaded. (B) 10µg of protein was loaded. White arrows indicate molecular weights in kDa. (C) NSF band intensity of fibronectin spread cells divided by control NSF band intensities within the whole cell lysate, cytoplasmic and nuclear cellular fractions. Bars represent quotient means determined from 3 experiments with standard deviations based on these means.

0.00!0.20!0.40!0.60!0.80!1.00!1.20!1.40!1.60!1.80!

Whole Cell Lysate!

Cytoplasmic! Nuclear!

Trea

ted/

Ctr

l NSF

ban

d in

tens

ity Q

uotie

nt!

Cellular Fraction!

A B!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!C!!!!!!!!!!!!!!!!

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showed that NSFE/Q mutants inhibited SNARE-mediated α5β1 integrin trafficking and

thereby cell spreading. Therefore, I sought to determine whether nuclear localization

could be determined using cellular fractionation and immunoblotting. CHO cells were

transferred to test plates coated with immobilized fibronectin and imaged 2-hours later.

Cell spreading was evident in cells plated on fibronectin when compared to cells

transferred to non-coated plates (Figure 10). Cellular fractionation and immunoblotting

showed an overall increase in whole cell NSF levels (quotient value of 1.21), with no

overall change to nuclear NSF levels (quotient value of 1.00) and a subtle decrease in

cytoplasmic levels (quotient value of 0.75) (Figure 11). This data verifies the previously

unreported nuclear localization of NSF, and corresponds well with the positive nuclear

control, TBP. Furthermore, although the data is not significant, the trend demonstrates

an upregulation of whole cell NSF levels during cell spreading.

3.3 Bioinformatics Analysis of NSF

Exportin 1 recognizes and binds to leucine-rich NESs, thereby facilitating nuclear

export. N-Ethylmaleimide Sensitive Factor has three putative NESs, with NES-1

containing two Leu and one Ile, NES-2 containing six Leu and NES-3 containing three

Leu and three Ile (Figure 1).

In order to better identify where the three NESs are found within the NSF

structure, I obtained an NSF structural model (residues 224-742) from the protein data

bank (PDB), and using RasMol highlighted the three regions of interest (Figures 12 and

13). Using MOLMOL I determined the percentage of the total surface area of each

residue that is solvent accessible, within each putative NES (Figure 12B, C, D).

Additionally, we have previously shown that point mutations of I705A and I707A in

NES-3 result in nuclear-NSF accumulation (Figure 7). Taken together, the data suggests

that NSF is shuttled out of the nucleus by Exportin 1.

3.4 NEM Affects Cellular Localization of NSF

When Block et al. (1988) treated Golgi networks of CHO cells with NEM, transport was

inhibited; it was this study that first led to the isolation of NSF (Block et al. 1988).

Consequently, we were interested in studying the effect of NEM on NSF localization,

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Figure 12. (A) N-ethylmaleimide Sensitive Factor (residues 224-742 at a resolution of 1.75Å) illustrating the three nuclear export sequences (NES) and (B, C, D) percentage of solvent accessible surface area. (A) The structural model was obtained from the protein data bank (Model ID: c1d6b18bb389199c6837f37889952f34) and using RasMol (version 2.7.5) the three putative NES were highlighted. (B, C, D) Using MOLMOL (version 2K.2) the percentage of the solvent accessible surface area of each residue was determined.

! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!B A ! !

!!!!!!!!!!!!!!!!!!!!!!!!! !!

C D

Amino Acid Number Amino Acid Solvent Accessible Surface (%) 600 VAL 11.1 601 VAL 1.7 602 VAL 11.0 603 ASP 9.8 604 ASP 19.5 605 ILE 16.5 606 GLU 21.5 607 ARG 34.3 608 LEU 8.8 609 LEU 18.2

!Amino Acid Number Amino Acid Solvent Accessible Surface (%)

621 LEU 42.5 622 VAL 22.6 623 LEU 5.5 624 GLN 11.8 625 ALA 13.8 626 LEU 46.4 627 LEU 18.2 628 VAL 23.3 629 LEU 23.2 630 LEU 44.6

!Amino Acid Number Amino Acid Solvent Accessible Surface (%)

705 ILE 31.9 706 GLY 13.5 707 ILE 39.0 708 LYS 42.6 709 LYS 33.5 710 LEU 8.4 711 LEU 32.0 712 MET 33.4 713 LEU 5.5 714 ILE 6.1

!

Amino Acid Number Amino Acid Solvent Accessible Surface (%) 600 VAL 11.1 601 VAL 1.7 602 VAL 11.0 603 ASP 9.8 604 ASP 19.5 605 ILE 16.5 606 GLU 21.5 607 ARG 34.3 608 LEU 8.8 609 LEU 18.2

!Amino Acid Number Amino Acid Solvent Accessible Surface (%)

621 LEU 42.5 622 VAL 22.6 623 LEU 5.5 624 GLN 11.8 625 ALA 13.8 626 LEU 46.4 627 LEU 18.2 628 VAL 23.3 629 LEU 23.2 630 LEU 44.6

!Amino Acid Number Amino Acid Solvent Accessible Surface (%)

705 ILE 31.9 706 GLY 13.5 707 ILE 39.0 708 LYS 42.6 709 LYS 33.5 710 LEU 8.4 711 LEU 32.0 712 MET 33.4 713 LEU 5.5 714 ILE 6.1

!

Amino Acid Number Amino Acid Solvent Accessible Surface (%) 600 VAL 11.1 601 VAL 1.7 602 VAL 11.0 603 ASP 9.8 604 ASP 19.5 605 ILE 16.5 606 GLU 21.5 607 ARG 34.3 608 LEU 8.8 609 LEU 18.2

!Amino Acid Number Amino Acid Solvent Accessible Surface (%)

621 LEU 42.5 622 VAL 22.6 623 LEU 5.5 624 GLN 11.8 625 ALA 13.8 626 LEU 46.4 627 LEU 18.2 628 VAL 23.3 629 LEU 23.2 630 LEU 44.6

!Amino Acid Number Amino Acid Solvent Accessible Surface (%)

705 ILE 31.9 706 GLY 13.5 707 ILE 39.0 708 LYS 42.6 709 LYS 33.5 710 LEU 8.4 711 LEU 32.0 712 MET 33.4 713 LEU 5.5 714 ILE 6.1

!

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Figure 13. (A) N-ethylmaleimide Sensitive Factor D2 monomer (residues 489-742 at a resolution of 1.75Å) illustrating the three nuclear export sequences (NES). The structural model was obtained from the protein data bank (Model ID: c1d6b18bb389199c6837f37889952f34) and using RasMol (version 2.7.5) the three putative NES were highlighted. (B) N-Ethylmaleimide Sensitive Factor D2 hexameric structure with boxed monomer in green reflecting D2 monomer orientation in (A). Image (B) was reprinted with permission (Yu, R.C. et al., 1998).

A B !!!!!!!!!!!!!!!!!! !

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specifically nuclear NSF localization. Furthermore, NEM has been shown to act as a

chromosome region maintenance 1 (CRM1 or Exportin 1) inhibitor by alkylating

Cys529 of Exportin 1 (Kudo, N. et al. 1999). Therefore, NEM treatment of CHO cells

was conducted to examine its effect on 1) NSF localization and 2) Exportin 1 activity.

In order to address these questions I examined nuclear NSF levels following cellular

fractionation and immunoblotting. Immunoblotting of NEM treated CHO cells showed

small overall changes in whole cell NSF levels, at all three NEM concentrations tested

(quotient values of: 0.99 at 0.04mM, 0.88 at 1.0mM and 1.02 at 5mM) (Figure 14A and

B). However, at a NEM concentration of 0.04mM, cytoplasmic and nuclear NSF levels

are higher than their respective control levels (quotient values of 1.10 and 1.20,

respectively). At 1mM NEM, cytoplasmic levels appear unchanged (quotient value of

1.01), while nuclear levels decrease (quotient value of 0.61). At 5mM NEM,

cytoplasmic levels show a subtle decrease (quotient value of 0.92), while nuclear levels

remain unchanged (quotient value of 1.01).

Although the data is not significant, the trend demonstrates that at high

concentrations of NEM whole cell, cytoplasmic and nuclear NSF levels remain largely

unchanged from control levels. At the lowest NEM concentration (0.04mM),

cytoplasmic and nuclear NSF levels increased, suggestive of Exportin 1 inhibition and

nuclear accumulation of NSF. However, the largest changes of whole cell and nuclear

NSF levels occur at a NEM concentration of 1mM, when NSF levels decreased (Figure

14B). These results are somewhat counterintuitive, suggesting an alternate route for NSF

export. N-ethylmaleimide appears to have an effect on nuclear NSF localization,

however the effect is not linear and requires further investigation in order to better

understand the mechanism in effect.

3.5 LMB Does Not Affect Nucleocytoplasmic Shuttling of NSF

Leptomycin B is used extensively in molecular biology as an inhibitor of Exportin 1.

Like NEM, it interferes with Exportin 1 activity by alkylating Cys529 (Kudo, N. et al.

1999). Therefore, I employed LMB treatment of CHO cells as a second method in

determining whether NSF depends on Exportin 1 for nuclear export. Imaging data

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Figure 14. Whole cell (W), cytoplasmic (C) and nuclear (N) fractions from NEM treated or not-treated CHO cells. Cells were incubated either in the absence or presence of NEM prior to fractionation. SDS-PAGE was performed followed by a western blot (A) 16µg of protein was loaded. White arrows indicate molecular weights in kDa. (B) NSF band intensity of 0.04mM, 1.0mM and 5mM n-ethylmaleimide (NEM) treated cells divided by control NSF band intensities in the whole cell lysate, cytoplasmic and nuclear cellular fractions. Bars represent quotient means determined from 3 experiments with standard deviations based on these means.

0!0.2!0.4!0.6!0.8!

1!1.2!1.4!1.6!

0.04! 1! 5!

Trea

ted/

Ctr

l NSF

ban

d in

tens

ity Q

uotie

nt!

Concentration of NEM (mM)!

Whole Cell Lysate!Cytoplasmic!Nuclear!

A !!!!!!!!!!!!!!B!

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Figure 15. Imaging of wtNSF-EGFP in CHO cells in the absence, presence and post-LMB (Xinping Qiu, unpublished data). CHO cells were treated with 200ng/mL of LMB and incubated for 4 hours. Cells were then washed, fixed and stained with DAPI so as to view the localization of wtNSF-EGFP with respect to the nucleus.

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showed a very subtle increase in nuclear localization of NSF during LMB treatment of

CHO cells when compared to controls (Figure 15; Xinping, Qiu, unpublished data).

Similarly, western blotting techniques indicated that at a LMB concentration of 1ng/mL,

whole cell and nuclear NSF levels showed a mild decrease from control levels (quotient

values of 0.90 and 0.92, respectively), while cytoplasmic levels increased (quotient

value of 1.21) (Figure 16A and C). At 10ng/mL of LMB whole cell NSF levels show a

strong increase (quotient value of 1.53), however cytoplasmic and nuclear NSF levels

are close to control values (quotient values of 1.05 and 0.94, respectively) (Figure 16A

and C). At 100ng/mL of LMB whole cell NSF levels remain unchanged (quotient value

of 0.97), however cytoplasmic NSF levels increased (quotient value of 1.30), while

nuclear NSF levels showed a decrease (quotient value of 0.60) when compared to

control levels (Figure 16A and D). With a two-fold increase in LMB concentration,

whole cell and cytoplasmic NSF levels increased (quotient values of 1.55 and 1.18,

respectively), while nuclear NSF levels remained unchanged from control levels

(quotient value of 0.99) (Figure 16B and D). A representative blot of all LMB

concentrations tested is shown (Figure 16A and B). LMB concentrations of 1ng/mL and

10ng/mL were only tested once. In summary, at 1ng/mL, 10ng/mL and 200ng/mL of

LMB, nuclear NSF levels are unchanged (quotient values approximately equaling 1.0),

however, at a LMB concentration of 100ng/mL nuclear NSF levels dropped. These

results in conjunction with the imaging data conducted by Xinping Qiu, imply that

inhibition of Exportin 1 by LMB does not interfere with nuclear export of NSF,

indicating that NSF does not rely on Exportin 1 for nucleocytoplasmic shuttling.

3.6 NSF Shows Nuclear Localization Across Several Cell Lines

In order to determine if NSF localizes to the nucleus in other cell lines, nuclear lysates

from four different cell lines were purchased. In addition to CHO cells, mouse

neuroblastoma (N2A) cells were cultured and cellular fractionation was performed.

Protein immunoblotting was performed using nuclear lysates derived from HeLa and

PC12 cells (Figure 17A). NSF was present in the PC12 nuclear lysate, though none was

detected in the nuclear fraction derived from HeLa cells (Figure 17A). Protein

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Figure 16. Whole cell (W), cytoplasmic (C) and nuclear (N) fractions from LMB treated or not-treated CHO cells. Cells were incubated either in the absence or presence of LMB for 4 hours, prior to fractionation. SDS-PAGE was performed followed by a western blot (A) 8.2µg of protein was loaded. (B) 8.0µg of protein was loaded. White arrows indicate molecular weights in kDa. (C) NSF band intensity of 1ng/mL and 10ng/mL LMB treated cells divided by control NSF band intensities in the whole cell, cytoplasmic and nuclear cellular fractions. (D) NSF band intensity of 100ng/mL and 200ng/mL LMB treated cells divided by control NSF band intensities in the whole cell, cytoplasmic and nuclear cellular fractions. Bars represent quotient means determined from 2 experiments with standard deviations based on these means.

0.00!

0.20!

0.40!

0.60!

0.80!

1.00!

1.20!

1.40!

1.60!

1.80!

2.00!

Whole Cell Lysate!

Cytoplasmic! Nuclear!Trea

ted/

Ctr

l NSF

ban

d in

tens

ity

Quo

tient!

Cellular Fraction!

LMB: 100ng/mL!LMB: 200ng/mL!

0!

0.2!

0.4!

0.6!

0.8!

1!

1.2!

1.4!

1.6!

1.8!

Whole Cell Lysate!

Cytoplasmic! Nuclear!Trea

ted/

Ctr

l NSF

ban

d in

tens

ity

Quo

tient!

Cellular Fraction!

LMB: 1ng/mL!LMB: 10ng/mL!

A

B C

D

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Figure 17. Immunostaining of NSF within nuclear lysate originating from several cell types. (A) HeLa and PC12 nuclear lysates; immunoblotting was conducted for the presence of NSF and TBP; tubulin served as a positive control (37.5µg of protein was loaded). (B) CHO, N2A, NIH3T3, PC12 and Jurkat nuclear lysates; immunoblotting was conducted for the presence of NSF and TBP; tubulin and actin served as a positive control (30µg of protein was loaded). SDS-PAGE was performed followed by a western blot. White arrows indicate molecular weights in kDa.

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immunoblotting was then performed using nuclear lysates derived from: CHO, N2A,

NIH 3T3, PC12 and Jurkat cells (Figure 17B). Cell lines showed varying levels of

nuclear NSF, with CHO cells showing the highest levels and Jurkat cells showing

absence of NSF (Figure 17B). It should be noted that nuclear lysates originating from

HeLa, PC12, NIH 3T3 and Jurkat cells were purchased, while nuclear lysates derived

from CHO and N2A cells were prepared using the Cellular Fractionation protocol.

These results confirm NSF nuclear localization in several cell lines and suggest that the

role nuclear NSF is executing is not exclusive to a specific cell type.

3.7 Nuclear NSF Interacts with Vimentin

In order to elucidate the nuclear function of NSF, I chose an immunoprecipitation

strategy to identify the binding partner(s) of nuclear-NSF. N-Ethylmaleimide Sensitive

Factor was immunoprecipitated from a CHO nuclear fraction and the resulting sample

was analyzed by mass spectrometry (Figure 18A). The mass spectrometry analysis

confirmed the presence of NSF in the sample and identified a number of co-

immunoprecipitated proteins (Figure 18B). Among these, the intermediate filament

protein Vimentin showed the highest number of unique peptides (31 unique peptides,

Figure 18C). In addition, several other interacting proteins were identified (for a sample

of co-immunoprecipitated proteins with a protein identification probability of >99.9%

see Figure 18D). Among these were exclusively nuclear proteins, histones H2A and H3,

and the cytoskeletal protein, actin (Figure 18D).

In order to confirm the NSF-Vimentin interaction the reverse IP approach was

taken using an anti-Vimentin antibody. Initially SyproRuby staining confirmed the

presence of protein in the nuclear IP sample and the absence of protein in the control

(Figure 19A). Immunoprecipitation from cytoplasmic and nuclear fractions was

performed, followed by immunoblotting (Figure 19B and C). Although NSF did not

immunoprecipitate with Vimentin from the cytoplasmic fraction, I did detect an NSF-

Vimentin interaction in the nuclear fraction (Figure 19B and C). These results confirm

the NSF-Vimentin interaction and suggest that the NSF-Vimentin interaction is

exclusive to the nucleus.

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Figure 18. N-Ethylmaleimide Sensitive Factor IP from CHO cells. (A) NSF IP from CHO cell nuclear fraction showing whole cell lysate (W), IP pull-down, flow through (FT), wash 1 (W1) and wash 3 (W3) samples in the presence (+) and/or absence (-) of the NSF antibody. SDS-PAGE was performed followed by a western blot. To confirm NSF presence immunoblotting was performed; the actin stain served as a positive control. White arrows indicate molecular weights in kDa. (B) NSF IP mass spectrometry results and (C) Vimentin IP mass spectrometry results. IP samples were sent to Sick Kids Proteomics Center for mass spectrometry analysis. Results confirmed the IP of NSF and revealed that Vimentin co-immunoprecipitated. Yellow highlights indicate 80-94% probability of residue identification and green highlights indicate over 95% probability of residue identification. (D) N-ethylmaleimide Sensitive Factor co-IP results as determined from mass spectrometry analysis conducted by Sick Kids Proteomics Center. All results show a >99.9% protein identification probability.

!

!

! ! ! !!!!!!!!!!A !!!!!!

!!! B C

D

Protein Name Accession Number

Molecular Weight

Protein Identification

Probability (%) Histone H2A type 1 344240017 28 100

Histone H3 344238671 30 100 Histone H3.1t 344242825 36 100

Vimentin 354482483 54 100 Actin, cytoplasmic 1 347360906 42 100

N-ethylmaleimide sensitive fusion protein 134267 83 100

78 kDa glucose-regulated protein

precursor 350537423 72 100

Stress-70 protein, mitochondrial 344250583 66 100

Granulins isoform 1 354484751 65 100

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Figure 19. Vimentin IP from CHO cells. (A) Vimentin IP from CHO cell nuclear fraction showing whole cell lysate (W), IP pull-down, flow through (FT) and wash 1 (W1) samples in the presence (+) or absence (-) of the Vimentin antibody. SDS-PAGE was performed followed by SYPRO Ruby staining. (B) Vimentin IP from CHO cell cytoplasmic and (C) nuclear fractions showing whole cell lysate (W), flow through (FT), wash 1 (W1) sample and IP pull-down samples in the presence (+) and absence (-) of the Vimentin antibody. SDS-PAGE was performed followed by a western blot. To confirm NSF and Vimentin’s presence immunoblotting was performed. White arrows indicate molecular weights in kDa.

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3.8 NSF is Present in the Nuclei of Prophase Cells Our unpublished data indicates that NSF nuclear dynamics may be associated

with the cell cycle, therefore, I sought to investigate the NSF nuclear profile in a

population of synchronized cells. Nocodazole is commonly used in molecular biology as

a tool for cell synchronization (Poxleitner, M.K. et al. 2008; Matsui, Y. et al. 2012).

Initially, to confirm that the NSF immunoprecipitated from the nuclear fraction was

truly nuclear, CHO cells were treated with nocodazole for 10-hours. This should have

arrested cells, however, cell synchronization was not achieved as only 20% of cells

reached chromosomal condensation, suggestive of prophase (Figure 20A).

As previously described, a cytoplasmic and nuclear Vimentin IP was performed

followed by immunoblotting (Figure 20B and C). As before, NSF was not associated

with the Vimentin IP from the cytoplasmic fraction, however both proteins were present

in the nuclear Vimentin IP (Figure 20B and C). These results confirm the nuclear NSF-

Vimentin interaction.

In order to ascertain that the identified nuclear NSF is truly nuclear, rather than

mistakenly present within the nuclei of newly formed cells still requiring reorganization,

it was necessary to perform nuclear localization prior to the disintegration of the nuclear

membrane. In order to accomplish this it was necessary to perform cellular fractionation

on early prophase cells. I, therefore, employed an alternate cell synchronization

protocol. Chinese hamster ovary cells were treated with thymidine for 24 hours. Upon

removal of thymidine, cells grown on test plates were fixed, stained with DAPI and

imaged at each hour, with time 0 marking the removal of thymidine, to a total of 18

hours (Figure 21). At 14.5 hours approximately 80% of the nuclei showed early

chromatin condensation, suggestive of early prophase (Figure 21B). At this point

cellular fractionation was performed followed by an NSF-IP of the cytoplasmic and

nuclear fractions in efforts to determine the localization of NSF during prophase (Figure

22). The IP results show that NSF is present in both the cytoplasmic and nuclear

fractions. It should be noted that the nuclear NSF band appears at a lower molecular

weight than the whole cell fraction NSF band (Figure 22B). This phenomenon is also

evident in Figures 11, 14 and 16. Likely these differences are due to post-translational

modifications of NSF, although further work is necessary to confirm this. However, this

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may prove to be a useful marker for distinguishing cytoplasmic and nuclear NSF in

future studies.

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Figure 20. Nocodazole mediated CHO cell synchronization. (A) DAPI staining of CHO cells after a 10-hour nocodazole (50ng/mL) treatment. Black arrow indicates a cell entering mitosis as evidenced by chromosomal condensation. (B) Vimentin IP from CHO cell cytoplasmic and (C) nuclear fractions following a 10 hour nocodazole (50ng/mL) treatment, showing whole cell lysate (W), IP pull-down, flow through (FT), wash 1 (W1) and wash 3 (W3) samples in the presence (+) and absence (-) of the Vimentin antibody. SDS-PAGE was performed followed by a western blot. To confirm NSF and Vimentin’s presence immunoblotting was performed. White arrows indicate molecular weights in kDa.

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Figure 21. Thymidine mediated CHO cell synchronization. Cells were incubated with 2nM thymidine for 24 hours. After incubation, thymidine was removed, cells were washed and their progression through the cell cycle was monitored for 18 hours, with T0 marking removal of thymidine, T1 marking one hour after thymidine removal and so forth. (A) Cells from T0 to T5 (B) from T6

to T12.5 and (C) from T13.5 to T18.

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Figure 22. N-Ethylmaleimide Sensitive Factor IP from thymidine synchronized CHO cells. (A) NSF-IP from the cytoplasmic and (B) nuclear fractions following a 24 hour thymidine (2mM) treatment, showing whole cell lysate (W), flow through (FT), wash 1 (W1), wash 3 (W3) and IP pull-down samples in the presence (+) and absence (-) of the NSF antibody. SDS-PAGE was performed followed by a western blot. To confirm NSF’s presence immunoblotting was performed. White arrows indicate molecular weights in kDa.

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4 Discussion

4.1 Overview

The present study was undertaken to investigate the nuclear localization of NSF and to

identify its nuclear binding partner(s) in efforts to characterize its nuclear role. I

hypothesized that NSF is a dynamic nuclear protein that interacts with other nuclear

proteins to control cell function. To test this hypothesis I first attempted to use different

means to bias nuclear-NSF localization: fibronectin induced CHO cell spreading and

drug induced Exportin 1 inhibition. To identify nuclear binding proteins, I

immunoprecipitated NSF and used mass spectrometry to identify any co-

immunoprecipitated products. I then tested nuclear-NSF localization within several cell

types. Lastly, I used drug-induced cell synchronization methods to identify nuclear NSF

localization at the subcellular level during specific cell cycle phases.

N-Ethylmaleimide Sensitive Factor whole cell and nuclear levels fluctuated

during cell spreading and NEM/LMB treatments. Immunoprecipitation identified NSF’s

putative nuclear binding as Vimentin and NSF was found to localize to the nucleus of

CHO, N2A, PC12 and NIH 3T3 cells.

Earlier data has shown localization of NSF to the nuclear membrane in PC12

cells (Tagaya, M. et al. 1996; Mashima, J. et al. 2000). Additionally, unlike golgi-

localized NSF, nuclear membrane localized NSF was not released when incubated with

Mg2+-ATP (Tagaya, M. et al. 1996; Mashima, J. et al. 2000). There also exists evidence

for NSF’s role in the formation of the nuclear envelope and assembly of the NPC (Baur,

T. et al. 2007). The current data surrounding the diverse and multiple roles played by

AAA+ proteins suggests that NSF is likely not limited to SNARE disassembly (for a

review of the AAA+ superfamily functions see: Snider, J. et al. 2008). In fact, there is a

strong line of evidence that shows NSF does play many roles and is not limited to the

cytoplasm (Tagaya, M. et al. 1996; Kamboj S.I. et al. 1998; Nishimune A. et al. 1998;

Osten P. et al. 1998; Mashima, J. et al. 2000; Matsushita, K. et al. 2003; Huang Y. et al.

2005; Baur, T. et al. 2007). My data supports this hypothesis and suggests of a novel

nuclear role for NSF.

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4.2 Bioinformatics Analysis of NSF

Three putative nuclear export sequences (NESs) were identified within the NSF D2-

domain (Figure 1). Blast sequence alignment of all three NESs showed conserved

hydrophobic residues between Homo sapiens, Cricetulus griseus, Drosophila

melanogastor, Dictyostelium discoideum and Saccharomyces cerevisiae (Figure 5).

Furthermore, hydrophobic residues at the specified positions show conservation between

NESs of different proteins (Figure 6). Identification of putative NESs facilitated studies

where point mutations were introduced into the putative NESs (Figure 7, Xinping, Qiu;

unpublished data). When two point mutations (I705A and I707A) were introduced into

the third NESs of mammalian NSF, transfected Schneider 2 cells of Drosophila

melanogaster showed a strong NSF signal within the nucleus. Taken together this data

strongly supports the correct identification of the NESs.

4.3 Fibronectin-Mediated Cell Spreading Affects NSF Localization

Fibronectin is a cell adhesion protein whose receptors are part of integrin-family of

proteins (Wennerberg, K. et al. 1997). Spreading of CHO cells plated on immobilized

fibronectin coated test-plates was evident after 2 hours (Figure 10). Immunoblotting data

revealed elevated whole cell NSF levels, although nuclear NSF levels did not show an

increase (Figure 11C). Cytoplasmic NSF levels, did however, show a decrease (Figure

11C). The increase in whole cell NSF levels should be accompanied with an increase in

nuclear and/or cytoplasmic NSF levels, however, the data does not show this. The loss

of sample is inevitable during the purification of the cytoplasmic and nuclear fractions;

these steps are omitted when preparing the whole cell fraction. It is the loss of sample

that likely gave rise to the incongruity in the results.

Nevertheless, when considering the whole cell fraction the data suggests that

during integrin activation, when much cytoskeletal reconstruction occurs within the cell,

NSF is upregulated within the cell. This is in agreement with other studies that found the

E329Q-NSF mutant to be an inhibitor of SNARE-mediated α5β1 integrin trafficking and

consequently cell spreading (Tayeb, M. A. et al. 2005; Skalski, M. and Coppolino, M.

G., 2005; Skalski, M. et al. 2011).

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4.4 NEM Affects the Subcellular Localization of NSF

N-ethylmaleimide treatment revealed an increase in nuclear-NSF at a NEM

concentration of 0.04mM, suggestive of Exportin 1 inhibition by NEM (Figure 14A and

B). Inhibiting nuclear export via leptomycin B results in the nuclear accumulation of

tubulin due to Exportin 1 inhibiton; NEM has been shown to inhibit nucleocytoplasmic

shuttling by the same mechanism (Kudo, N., 1999; Akoumianaki, T. et al. 2009).

Consequently, tubulin was used as a positive control for both NEM and LMB

treatments. Nuclear tubulin increased at 0.04mM NEM (Figure 14A). However, there is

a decrease in nuclear NSF and tubulin levels at a NEM concentration of 1mM (Figure

14A and B). This result is counterintuitive, as one would expect an increase in nuclear

accumulation of both proteins with an increase in Exportin 1 inhibition. Interestingly, at

5mM NEM whole cell, cytoplasmic and nuclear NSF levels return to control levels. It

may be speculated that the decrease of nuclear NSF and tubulin at 1mM NEM, despite

the increase at 0.04mM NEM, is due to an alternate export machinery.

4.5 Exportin 1 is Likely Not Responsible for Nuclear-NSF Export

Imaging data of CHO cells treated with LMB and protein immunoblotting revealed a

very mild increase in the nuclear localization of NSF (Figure 15 and 16). These results

are comparable to those seen with the NEM treatments (Figure 14). The data at 1mM

NEM and 100ng/mL LMB implies that Exportin 1, which has been classified as an

exportin that shuttles leucine rich NES cargo, may not be responsible for nuclear export

of NSF (Pemberton, L.F. et al. 2005). Alternatively, NSF may be shuttled out of the

nucleus by Exportin 1, but not depend on the LMB sensitive motif of Exportin 1 for this

transport (Connor, M.K., et al. 2003). To further investigate whether the mild effect of

NEM and LMB on NSF is due to an alternate nuclear export route, imaging analysis of

NSF-NES mutants may be used. NSF-NES mutants with point mutations introduced at

positions of leucine residues could be imaged for nuclear NSF accumulation, as a

function of time, during NEM/LMB treatment in order to monitor nuclear NSF

accumulation with Exportin 1 inhibition. Alternatively, the putative NESs of NSF may

be tagged onto a green fluorescence protein (GFP) and microinjected into the nuclei of

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CHO cells. Cells may be treated with NEM and LMB and the localization of the NES-

tagged GFP monitored. This experiment would help to 1) further validate the correct

identification of the NESs and 2) ascertain whether Exportin 1 is responsible for NSF

export.

4.6 Evidence for Cytoplasmic Post-Translational Modification of NSF

It is quite evident that nuclear-NSF resolves at a slightly lower molecular weight than

cytoplasmic-NSF (Figures 11, 14 and 16). This phenomenon may be due to

posttranslational modifications of NSF such as S-nitrosylation or phosphorylation. S-

nitrosylation of NSF has been reported as a means of regulation during AMPA receptor

recycling and during exocytosis of Weibel-Palade bodies (Huang, Y. et al., 2005;

Matsushita, K. et al. 2003). Phosphorylation of NSF by Pctaire1 on serine 569 in the D2

domain has been reported as a means of regulating NSF’s oligomerization (Liu, Y. et al.,

2006). While phosphorylation by protein kinase C (PKC) at serine 237 in the D1 domain

results in NSF’s inability to bind to the SNAP-SNARE complex (Matveeva E.A. et al.,

2001). Though it has been reported that the sulfur-nitric oxide bond is cleaved during

SDS-PAGE manipulation of a sample, a phosphate group remains and can be detected

on a western blot (Torta, F. et al., 2008; Kocinsky, H.S. et al., 2005). Further

experiments will have to be conducted to test whether the differences observed in the

way nuclear versus cytoplasmic-NSF resolve on the SDS-PAGE gel, relates to its

phosphorylation. If indeed, cytoplasmic and nuclear NSF resolve at distinct molecular

weights on account of post-translational modifications, this difference may be exploited

in distinguishing the levels of cytoplasmic versus nuclear NSF.

4.7 NSF Localizes to the Nuclei of Various Cell Lines & Cell Types

Immunoblotting revealed NSF is present in nuclear lysates originating from PC12,

CHO, N2A and NIH3T3 cell lines (Figure 17A and B). The nuclear lysate derived from

CHO cells had the highest level of nuclear NSF. An anti-TBP antibody was used as a

positive control, however the success of the stain varied across the cell lines. This is

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likely due to the fact that the host animal, mouse, will show better reactivity with mouse,

rat and hamster-originating samples than human samples, and this is indeed the result.

Nonetheless, TBP is present across all nuclear samples tested.

The presence of NSF in nuclear lysates of other cells is an important contribution

to the current study as it demonstrates NSF’s nuclear localization between species and

across a range of cell lines. A previous report on the nuclear localization of NSF by

Tagaya, M. et al. (1996) demonstrated the presence of NSF in the nuclear membrane of

PC12 cells. The authors confirmed that the nuclear membrane-associating NSF was not

released when treated with Mg2+-ATP, unlike the golgi-associating NSF, which was

released (Tagaya, M. et al. 1996; Mashima, J. et al. 2000). Additionally, the molecular

weight of the resolved NSF bands varies between the cell lines tested (Figure 17B). The

CHO, N2A, NIH 3T3 and PC12 nuclear lysates all appear to have two bands detected by

the NSF antibody, one appearing at approximately 78kDa and the other at 70kDa

(Figure 17B). These differences between samples may result from differences between

the cell lines themselves. Because most of the cell lines tested have differentiated (NIH

3T3 cells differentiate further), their roles within an organism vary, and the levels of

nuclear versus cytoplasmic NSF may, consequently, also vary (Wang, Z. et al. 2011).

Further studies will have to investigate the level of nuclear NSF localization within a

range of cell lines, as well as the differences in molecular weights, as our data suggests

these factors may be variable.

4.8 Vimentin: A Putative Nuclear Binding Protein of NSF

Once nuclear localization of NSF was confirmed, and the extent of its sensitivity to

NEM and LMB treatments within the nuclei of CHO cells was demonstrated, IP of

nuclear NSF was employed in order to determine if there are nuclear-specific binding

partner(s) (Figure 18A). Mass spectrometry analysis of IP samples was conducted by the

Sick Kids center for proteomics (Figure 18B and C). Mass spectrometry analysis

confirmed the IP of nuclear NSF and identified several proteins that co-

immunoprecipitated (Figure 18B and D). Among those identified, the intermediate

filament, Vimentin, had the highest number of unique peptides (Figure 18C). I

confirmed the NSF-Vimentin interaction using a reverse IP approach (Figure 19).

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Because I compared co-IP of NSF and Vimentin in cytoplasmic and nuclear fractions,

and only found an interaction in the nuclear fraction, my data strongly suggest that the

NSF-Vimentin interaction is restricted to the nucleus.

Vimentin is a member of the group of proteins that make up intermediate

filaments. Intermediate filaments are a family of close to 70 proteins, divided into six

groups based on their sequence similarity, which fall intermediate in diameter size (10

nm) when compared to actin filaments (7 nm) and microtubules (25 nm) (Cooper, G.M.

2000; Satelli, A. and Li, S. 2011). Intermediate filaments are not as dynamic in their

assembly and disassembly as actin filaments and microtubules (Cooper, G.M. 2000).

Additionally, unlike actin filaments and microtubules, they are not responsible for cell

movement but serve to provide structural support to the cell, and phosphorylation of

intermediate filaments affects their assembly and organization (Cooper, G.M. 2000).

Within the family of intermediate filaments the six classes show differences in

expression and in intracellular organization. For example, type I and II intermediate

filaments form only heterodimers, while type III intermediate filaments may form

homodimers or heterodimers (Cooper, G.M. 2000; Satelli, A. and Li, S. 2011).

Vimentin is a 57-kDa, highly conserved, type III intermediate filament and it is

often used as a mesenchymal cell marker (Wu, Y. et al. 2007). Additionally,

overexpression of vimentin is correlated with poor prognosis in cancer patients (Satelli,

A. and Li, S. 2011). Vimentin shows differential expression in several cell lines due to

the varying roles it plays (Satelli, A. and Li, S. 2011). Like all intermediate filaments it

is apolar, with a central α-helix, necessary for intermediate filament assembly (Cooper,

G.M. 2000).

Vimentin forms polymers, either with itself or with other type III/IV intermediate

filaments (Cooper, G.M. 2000; Satelli, A. and Li, S. 2011). This polymerization is

facilitated by the conserved central α-helix and is common within the intermediate

filament proteins (Cooper, G.M. 2000; Satelli, A. and Li, S. 2011). During

polymerization, two Vimentin polypeptides align in parallel and their α-helices

intertwine, this dimer associates with a second Vimentin dimer in an anti-parallel

orientation forming an apolar tetramer (Cooper, G.M. 2000). The linear alignment of

several tetramers forms an apolar protofilament (Cooper, G.M. 2000). The regulation of

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assembly, disassembly, stability and activity of Vimentin and intermediate filaments in

general is dictated by post-translational modifications (Satelli, A. and Li, S. 2011).

Vimentin has been shown to undergo phosphorylation, SUMOylation, citrullination and

O-GlcNAcylation (Cheng, T.J. et al., 2003; Goto, H. et al., 2002; Liming Wang, J.Z. et

al., 2010; van Venrooij, W.J. and Pruijn, G.J., 2000; Vossenaar, E.R. et al., 2004;

Farach, A.M. and Galileo, D.S., 2008).

Initially, Vimentin was known for its cytoplasmic roles and thought to reside in

the cytoplasm. However, when fluorescently labeled Vimentin was coinjected with

single-stranded DNA and circular DNA into the cytoplasm of cultured cells it localized

to the nucleus (Hartig, R., et al., 1998). Recently, Vimentin has been shown to play a

nuclear role in the regulation of p21Waf1 expression in neuroblastoma cells, though the

mechanism by which it operates is still not well understood (Mergui, X., et al., 2010).

Additionally it has been shown to reduce activating transcription factor 4 (ATF4)

activity, thereby inhibiting osteocalcin transcription (Lian, N., et al., 2009). Vimentin’s

diverse cytoplasmic roles suggest its nuclear roles will reflect the same diversity and

vary from cell-line to cell-line, however much more investigation is required.

Vimentin has been linked to several cancers, such as prostate, breast and

gastrointestinal tract cancers just to name a few, and its expression pattern varies among

them (Lang, S.H. et al., 2002; Hu, L. et al., 2004; Gilles, C. et al., 2003). However, in

all cancers its over-expression is associated with poor prognosis due to the metastatic

and invasive phenotype of the resulting cancer cells (Cooper, G.M. 2000; Satelli, A. and

Li, S. 2011). The mechanism by which Vimentin facilitates these phenotypes is still

under investigation (Satelli, A. and Li, S. 2011). Despite this, it is becoming apparent

that Vimentin may prove to be a marker for cancer prognosis and a possible target for

future chemotherapeutic agents.

4.9 NSF is Present in the Nuclei of Prophase Cells

Nocodazole is used extensively in cell synchronization protocols in efforts to bring a

pool of cells into mitosis but arrest them at metaphase (Harper, J. V. 2005; Matsui, Y. et

al. 2012). Upon removal of nocodazole, cells should continue to progress normally

through the cell cycle (Harper, J. V. 2005). Correspondingly, nocodazole treatment was

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used in efforts to synchronize the cell cycle and to rule out the possibility that the

detected nuclear-NSF was not truly cytoplasmic NSF that had not been shuttled out of a

newly formed nucleus of a G1 cell. However, upon treatment it is difficult to say

whether cell synchronization was achieved (Figure 20A). Nocodazole treatment of CHO

cells resulted in approximately 20% of cells reaching chromosomal condensation,

suggestive of prophase (Figure 20A). Therefore, cell synchronization was not achieved.

Nonetheless, an IP of Vimentin from the nocodazole treated cytoplasmic and

nuclear fraction was performed, and an NSF signal, albeit weak, was detected in the

nuclear IP sample (Figure 20B and C). No NSF signal was detected in the cytoplasmic

IP fraction (Figure 20B). These results are in congruence with the Vimentin IP results of

nocodazole untreated CHO cells (Figure 19). However, it should be noted that studies

have shown that although nocodazole disrupts microtubule formation, it does not arrest

cells in a single stage of the cell cycle (Cooper, S. et al. 2006).

Other forms of cell synchronization that have been shown to be effective with

CHO cells involve treatment with DMSO (Fiore, M. et al. 2002). Fiore, M. et al. (2002)

found that CHO cells grown with 1-2% DMSO for 4 days were synchronized at the G1

phase. Thymidine is also added to cell cultures as a means to prevent nucleotide

synthesis and consequently, DNA synthesis, arresting cells in the S phase of interphase

(Jackman, J. and O’Connor, P. 2001; Harper, J. V. 2005). Therefore, to further confirm

the above results, a thymidine block was used to arrest CHO cells at the S-phase (Figure

21).

After removal of thymidine cells were monitored at each hour until early prophase

was reached by approximately 80% of cells (Figure 21C, T14.5). At this point cellular

fractionation was performed followed by immunoprecipitation. Immunostaining

revealed that NSF is present in the cytoplasm and nuclear fractions of prophase cells

(Figure 22). Therefore, because the Vimentin-IP revealed an absence of NSF in the

cytoplasmic fraction, but presence of NSF in the corresponding nuclear fraction, the data

suggests that the NSF-Vimentin interaction is exclusively nuclear (Figure 19B and C).

Taken together, this data indicates that the NSF immunoprecipitated in this and earlier

experiments is truly nuclear.

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5 Summary and Conclusion Recent studies have shown the nuclear effects of Vimentin include regulation of

p21Waf1 expression in neuroblastoma cells and down-regulating the activity of

activating transcription factor 4 (ATF4) resulting in the inhibition of osteocalcin

transcription (Mergui, X., et al., 2010; Lian, N., et al., 2009). The over-expression of

Vimentin has been linked with poor prognosis in several cancers (Cooper, G.M. 2000;

Satelli, A. and Li, S. 2011). The resulting cancer cells tend to exhibit invasive and

metastatic behaviors (Cooper, G.M. 2000; Satelli, A. and Li, S. 2011). Unfortunately,

the pathway leading to this phenotype is still not understood (Satelli, A. and Li, S.

2011). Although more work is required to confirm NSF’s nuclear role and its nuclear

interaction with Vimentin, it is becoming apparent that Vimentin, and possibly NSF,

may prove to be markers for cancer prognosis and possible targets for future

chemotherapeutic agents. Considering NSF’s well-studied role as a chaperone protein,

serving to dissociate the SNARE complex, I speculate that NSF’s nuclear role may

involve the disassembly of a complex, which Vimentin associates with.

It is worth contemplating why earlier studies have not detected nuclear-NSF.

Searching through the literature provides some answers to this conundrum. Although,

there are studies that examined imaging and co-locolazation of NSF with several other

proteins, no study to current date has examined nuclear localization of NSF and no study

conducted cellular fractionation followed by immunoblotting to test for the presence of

NSF within the nucleus (Song, I. et al. 1998; Mukhopadhyay, S. et al. 2008; Zhao, C. et

al. 2010). Imaging of NSF will not reveal nuclear-NSF, as shown in our results (Figures

7 and 15). This is likely due to the levels of nuclear-NSF when compared to those of

cytoplasmic-NSF. Without a doubt, there is a higher concentration of NSF within the

cytoplasm than that within the nucleoplasm. This surely reduced the chances of nuclear-

NSF detection and consequently, cellular fractionation and immunoblotting was not

pursued.

In conclusion, using immunoblotting techniques I have shown that NSF localizes

to the nucleus of several cell lines and using CHO cells I have demonstrated that its

nuclear levels change in the presence of LMB and NEM and during fibronectin-

mediated cell spreading. Immunoprecipitation and reverse co-IP of nuclear-NSF

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facilitated the identification of its putative nuclear binding partner, Vimentin. I have

shown preliminary data that suggests that this interaction is exclusive to the nucleus.

Additionally, I have shown that nuclear NSF is present in early prophase cells, ones with

intact nuclear membranes, arguing against cytoplasmic NSF contamination. Determining

the nature of the nuclear NSF-Vimentin interaction and its function will lead to the

characterization of a novel role for NSF. Ultimately, this mechanism will add to the list

of functions NSF and other AAA+ ATPases are responsible for.

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Appendix A

Recipe of Necessary Buffers for Cellular Fractionation Protocol as per Lamond Lab (University of Dundee, Dundee, Scotland)

* All tables were adapted from:

http://greproteomics.lifesci.dundee.ac.uk/webpage%20front%20page/dreamweaver%20

webpage/CellFractionation.pdf.