Using Drosophila to Study the PINK1-Parkin Mitochondrial
Quality Control PathwayLeo Pallanck
University of Washington
MitofusinsDrp1
The PINK1-Parkin PathwayMitofusins
ParkinPINK1
Lysosome
MitofusinsDrp1
The PINK1-Parkin PathwayMitofusins
ParkinPINK1
Contributions of fly genetics to this model:=>PINK1 & Parkin mutants accumulate enlarged defective mitochondria=>PINK1 & Parkin act in a common pathway=>Parkin ubiquitinates mitofusin to promote mt fragmentation
The PINK1-Parkin Pathway
Lysosome
MitofusinsDrp1
Mitofusins
ParkinPINK1
Some important remaining questions:
=>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons?
=>Do PINK1 & Parkin promote mitochondrial turnover in vivo?
=>What other factors act in the PINK1-Parkin pathway?
The PINK1-Parkin Pathway
Lysosome
MitofusinsDrp1
Mitofusins
ParkinPINK1
Our approach: stable isotope labeling and mass spectrometry
(collaboration with the laboratory of Mike MacCoss)
D3D3
D3 D3D3
time
trypsin digest total
proteins 5 & 10 days
CD3
D3-leucine (mass:
+3)
% o
f leu
cine
in p
eptid
es
D3-leucine
unlabeled leucine
Do PINK1 & Parkin promote mitochondrial turnover in vivo?
Technical challenges with this approach:
Solution: TOPOGRAPH; an algorithm that we helped developed in collaboration with the MacCoss laboratory
2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover
1. Quantifying the amount of D3-leucine incorporation into protein
actual amount of isotope
% 0 D3-leucines% 1 D3-leucine
isotope (additional mass units)
Frac
tion
al
Abun
danc
e
predicted
1. Quantifying the amount of D3-leucine incorporation into proteinTopograph finds the “best fit” (natural isotope distribution + n D3-leucines)
2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover
1. Quantifying the amount of D3-leucine incorporation into protein
Technical challenges with this approach:
Solution: TOPOGRAPH; an algorithm that we developed in collaboration with the MacCoss laboratory
D3-leucine content after
X hours of labeling: 25%
e.g., AIGLPEDLIQK (2 leucines)
Requires multiple Leu containing
peptides
precursor pool100% D3-leucine
precursor pool 50% D3-
leucine
25% turnover
2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover
50% turnover
“Old”
“New”
“Old”
“New”
TOPOGRAPH determines
this distribution
= 50% turnover
and uses probability-based calculations to estimate the D3-leucine content of the precursor pool
enables an estimate of turnover
“New”:
“Old”:
2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover
control
time
% D
3-le
ucin
e
Mitochondrial proteins
PINK1 or parkin mutants
Predictions of the PINK1-Parkin mitochondrial QC
model:
PINK1 or Parkin overexpression
control
time
% D
3-le
ucin
e
Non-mitochondrial
proteins
PINK1 or parkin mutants
PINK1 or Parkin overexpression
mitochondrial protein turnover is decreased in parkin mutants
0
200
400
600
800
1000
1200
1400
controlparkin–/–
half-
life
(hou
rs)
cont
rol
parki
n–/–
0
100
200
300
400
500
600
700
800
900
mea
n ha
lf-lif
e (h
ours
)
*p<0.0001
mean decrease in half-life: ~31%
nonmitochondrial protein turnover is similarly decreased in parkin
mutants
0
2
4
6
8
10
12
controlparkin–/–
half-
life
(hou
rs)
cont
rol
parki
n–/–
0
50
100
150
200
250
300
350
400
450
500
mea
n ha
lf-lif
e (h
ours
) *p<0.02
Why are nonmitochondrial proteins affected?
• Parkin regulates protein turnover more broadly than anticipated?
• The accumulation of defective mitochondria has a general effect on mitochondrial turnover?
0
100
200
300
400
500
600
700
800
controlPINK1 OE
half-
life
(hou
rs)
cont
rol
PINK1 O
E0
100
200
300
400
500
600
mea
n ha
lf-lif
e (h
ours
)
mean decrease in half-life: ~10%
*p<0.0001
PINK1 overexpression increases mitochondrial protein turnover
Importantly, mitochondrial proteins are selectively affected by PINK1
overexpression
0
2
4
6
8
10
12
mea
n %
cha
nge
(PIN
K1
OE
vs. c
ontr
ol)
mitochondrial nonmitochon-drial
*p < 0.0001
Important remaining questions:
=>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons?
=>Do PINK1 & Parkin promote mitochondrial turnover in vivo?
=>What other factors act in the PINK1-Parkin pathway?
The PINK1-Parkin Pathway
Lysosome
MitofusinsDrp1
Mitofusins
ParkinPINK1
Do PINK1 & Parkin influence mitochondrial QC in dopamine
neurons?First needed to develop a simple method to purify specific dopaminergic neurons from the fly brain:
-Uses the UAS/GAL4 system to mark the neurons of interest
(Brand and Perrimon, 1993)
GFP
ExamplesDopaminergic neuronsCholinergic neuronsEtc.
-Uses FACS to collect the marked neurons following mechanical and enzymatic brain dissociation
Non-transgenic flies
DAP
I
FITC (GFP)
TH-GAL4; UAS-GFP
FITC (GFP)
FACS purification of dopaminergic neurons from the adult Drosophila brain:
DAP
I
2% of events 0.02% of events
FACS-purified dopaminergic neurons also express appropriate markers (e.g., TH, VMAT, etc.)
Predictions:-mt membrane potential should correlate with PINK1-Parkin activity-accumulation of enlarged mt in parkin mutants.
+CCCP
-CCCP
Mitotracker fluorescence
# of
cel
ls#
of c
ells 78%22%
0%100%
55%45%
DA neurons DA neuronspark+/-
park-/-
38%62%
Rela
tive
MM
P
WT Park-/- PINK1OE
1.20.80.4
0
P < 0.01
P < 0.05
% fu
sed
mt
WT Park-/-
10080
40
0
P < 0.01
Mitotracker fluorescence
20
60
Do manipulations that promote mt fragmentation and turnover influence the
parkin neuronal phenotypes?
WT
0.40
0.8
park-/-
TgDRP1park-/-
Mfn Rnai
park-/-
TgAtg8a
1.2
park-/-
P < 0.01
Lysosome
MitofusinsDrp1
Mitofusins
PINK1Re
lativ
e M
MP
WT
0.40
0.8
park-/-
TgDRP1park-/-
Mfn Rnai
park-/-
TgAtg8a
1.2
park-/-
P < 0.001
Rela
tive
#PP
L1 n
euro
ns
-An enhanced sensitivity of mitochondria?-An increased sensitivity of cell survival in response to a general mitochondrial defect?
What explains the selective sensitivity of dopaminergic neurons to mutations in PINK1
and parkin?Re
lativ
e M
MP
WT DA neuronsPark-/-
1.20.80.4
0
P < 0.01
P < 0.05
% fu
sed
mt
WT Park-/-
10080
40
0
P < 0.01
20
60
=>Mitochondria in dopaminergic neurons are more sensitive to perturbations of the PINK1-Parkin pathway
Cholinergic neurons
Park-/-
Important remaining questions:
=>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons?
=>Do PINK1 & Parkin promote mitochondrial turnover in vivo?
=>What other factors act in the PINK1-Parkin pathway?
The PINK1-Parkin Pathway
Lysosome
MitofusinsDrp1
Mitofusins
ParkinPINK1
UAS-PINK1ey-GAL4
Cy
Del
Sb
++
++
; ;X
UAS-PINK1ey-GAL4
+;
Del
+suppression or enhancement
?
A screen for PINK1 overexpression modifiers:
WT
PINK1
PINK1park-/-
A deletion bearing chromosome
Will this work???
Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway?-mutations of activators (like parkin) should suppress-mutations of inhibitors (like mitofusin) should enhance
A screen for PINK1 overexpression modifiers:
WT
PINK1
PINK1park-/-
Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway?-mutations of activators (like parkin) should suppress-mutations of inhibitors (like mitofusin) should enhance
Will this work???
Eye severity
score
WT PINK1OE
24
0
6
PINK1OE
Park-/-PINK1OE
Park+/-PINK1OE
Mfn+/-
A screen for PINK1 overexpression modifiers:
WT
PINK1
PINK1park-/-
Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway?-mutations of activators (like parkin) should suppress-mutations of inhibitors (like mitofusin) should enhance
Will this work???
Eye severity
score
WT PINK1OE
24
0
6
PINK1OE
Park-/-PINK1OE
Park+/-PINK1OE
Mfn+/-
Suppressor
Enhancer
A screen for PINK1 overexpression modifiers:
-We have thus far screened deletions covering >95% of the genes residing on two of the three major chromosomes in Drosophila31 suppressors53 enhancersSome suppressors: Parkin, p62Some enhancers: mitofusin, Afg3L2Ongoing efforts:
Some are stronger modifiers than Parkin and Mitofusin
-Testing the specificity of modifiers
-mapping the modifier gene
Summary-The PINK1-Parkin pathway promotes selective mitochondrial turnover in vivo
-PINK1 & Parkin influence mitochondrial QC in dopaminergic neurons
-Mitochondria in dopaminergic neurons are selectively sensitive to the loss of the PINK1-Parkin pathway (a toxic effect of dopamine?)
-We’ve identified a large collection of candidate PINK1-Parkin pathway components
AcknowledgementsEvvie VincowJonathon BurmanRuth ThomasMichael MacCossNick ShulmanDepartment of Genome SciencesUniversity of Washington
Former Contributors:Angela PooleCornell UniversityAlex WhitworthUniversity of SheffieldJessica GreeneFred Hutchinson Cancer Research Center
National Institutes of HealthUMDF
How accurately does Topograph estimate the
precursor pool?-A validation experiment using yeast
1. Uniformly label yeast proteins for many generations in media containing a defined D3-leucine content
2. Subject the yeast proteins to mass spectrometry and use Topograph to measure the precursor poolAmount of D3-
Leucine in media33%67%
Topograph’s estimate of D3-Leucine precursor
pool33%68%
Pretty Accurately!!!