thesis research
TRANSCRIPT
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Cellular Role of the Drosophila EFR3 Homolog Rolling Blackout (RBO) in Synaptic Transmission
Niranjana Vijayakrishnan
Dissertation Defense, March 10th, 2010
Broadie Lab
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Why bother with Drosophila?
http://www.ipmc.cnrs.fr/~duprat/neurophysiology/brain.htm
http://www.sciencemag.org/sciext/vis2005/show/images/slide1_large.jpg
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Forward Genetics
Mutagenize
Screen for conditional temperature-sensitive (TS)
paralysis
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Synaptic transmission
paralytic : A subunit of Na+ channelVoltage Gated Na+ channel
Action Potential, initiation, propagation
Exocytosissyx3-69
Endocytosisshibire, GTPase dynamin
Where does Rolling Blackout (RBO) function?
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Temperature-sensitive paralysis in rbots
Flies @37OC: Selected time points from 0-6 minutes of heat shock
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ROLLING BLACKOUT (RBO)
•Essential gene, encodes an integral membrane protein predicted to be a lipase
•Conditional temperature-sensitive (TS) paralyticParalysis temperature 37oC
•Cloned and mapped in the Broadie Lab in 2001 (Huang et al., Nature Neuroscience , 2004)
•G527D missense mutation causes TS phenotype
•Homologs from yeast to humans
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Rolling Blackout: Protein Localization in Larval Synapses
Central Nervous System
rbo EGFPTransgenic fly line
Neuromuscular Junction
Huang et al., Nature Neuroscience, 2004 Vijayakrishnan and Broadie, Biochem soc Trans, 2006
SYT BRP
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rbots: Neurotransmission Defect
rbots WT
Dorsal longitudinal flight muscle (DLM)
Stimulate
Record
Interneuron
Huang et al., The Journal of Neuroscience, March 1, 2006
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rbots:Synapse Ultrastructure
# of
doc
ked
vesi
cles
per
acti
ve zo
ne
22 C 37 C
WT
rbo
rbo
rbots at 37 C: docked vesicles
Huang et al., The Journal of Neuroscience, March 1, 2006
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Genetic interaction with syntaxints
Richmond and Broadie, Curr Opin Neurobiol 2002 , 12, 499-507
Huang et al., The Journal of Neuroscience, March 1, 2006
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G protein coupled Receptor
PLC= Phospholipase C b
PIP2 DAG+ IP3
??Na+ and
Ca2+
bg Ga
TRP
PLC
DAG=DiacylglycerolPIP2= Phosphotidylinositiol (4,5) bis phosphate
TRP= Transient Receptor Potential
Drosophila Vision Cascade
Light
Garcia-Murillas et al., Neuron 49, 533–546, February 16, 2006
Complete loss of light-dependent receptor potential
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Glycine to Aspartic acid
Temperature–sensitive site
GXSXG
Aspartate/Glutamate
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RBO a lipase?
H S G-D D0 289 358 527 719
G-X- -X-G
834
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rolling blackout ts (rbo) Phenotype : Summary
Temperature-sensitive conditional paralytic and blind mutant Protein localized to fly nervous system- CNS and PNS Loss of EJC response from adult fly DLM at restrictive temperatureUltrastructure: Vesicle accumulation in DLM boutons Increase in vesicles at active zone (docked vesicles)Synergistic genetic interaction with t-SNARE Syntaxin 1A- rbots; syxts mutants
paralyze at 33oC
Homology to known lipasesHPTLC analysis of lipids revealed an increase in overall PIP and PI(4,5)P2 levels
and a decrease in DAG levels.
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xProposed Model for RBO Function
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1) Activity –dependent loading
Exocytosis Endocytosis
Assay for imaging synaptic vesicle cycling: FM dyes
FM1-43
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2) Destaining of loaded dye-assay for exocytosis
Assay for imaging synaptic vesicle cycling: FM dyes
EndocytosisExocytosis
FM1-43
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Drosophila 3rd instar larval neuromuscular junction (NMJ)
7 6 13 12
4
8
5
a
p
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Protocol 1
37 CO
25 CO
25 CO
syx
rbo
rbo;syx
25 C Load 37 C UnloadO O
OR
syx
rbo
rbo;syx
Depolarization-dependent FM1-43 Dye Loading: Assay Vesicle Exocytosis
60 mM K+
FM1-43Imaging
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FM1-43 Dye Loading Defects in rbots
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25 CO
25 CO
37 CO
Protocol 2
60 mM K+
FM1-43Imaging
rbo;syx
rbo
syx
OR
O O
37 C Load 25 C Unload
Depolarization-dependent FM1-43 Dye Loading: Assay Vesicle Endocytosis
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0
10
20
30
40
50
60
70
80
90
100
L L L L L L L LUL UL UL UL UL UL UL ULOR ORsyx syxrbo rborbo;syx rbo;syx
Mea
n Fl
uore
scen
ce In
tens
ity
Protocol 1
p<.001
p<.001p<.001
37 oC25 oC
Protocol 2
FM1-43 Dye Loading Defects in rbots
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0
20
40
60
80
100
OR rbo Rescue
Fluo
resc
ence
Inte
nsity
(AU
)
wt rbo-eGFP in rbo ts/D
Endocytic defects in rbots mutants are rescued by the wild-type rbo gene
rbo Rescue 25 CO
37 CO
25 CO
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RBOEGFP Expression in Cultured Pupal Neurons
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RBO localizes to functional synapses with cycling synaptic vesicles
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RBO facilitates endocytosis in central brain synapses
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rbo-EGFP
rbo mutants show defects in tracer uptake in Garland cells
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Garland cell UltrastructureB
C
37o C
37o C
OR rbots
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Num
ber o
f End
osom
es/s
ectio
n
Ultrastructure: rbo mutants show defects in tracer uptake in Garland cells
OR rbo
Block in Horse Raddish Peroxidase uptake into endosome at 37oC in rbo
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OR rbo
37o C
37o C
10
min
Hi [
K+ ]
Cist
erna
e N
umbe
r
Rest Stim
Ultrastructure: rbo mutants show defects in Endosome/cisternae formation at the NMJ
OR rbots
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Ultrastructure: rbo mutants show defects in FM1-43 uptake into Endosomes/cisternae at the NMJ
OR rbo
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Ultrastructure: rbo mutants show defects in FM1-43 uptake into Endosomes/cisternae at the NMJ
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Why does the loss of function syx3-69 mutation exacerbate the rbots phenotype at the NMJ?
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Lagow et al, 2007, PLOS Biology
syx3-69 loss of function allele?
Synaptic transmission persists at 38oC in syx3-69 in neurons in the eye and the flight muscle
•At 25oC: increase in “mini” frequency and amplitude of evoked release
•Mutation T254I: Dominant positive effect
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Conclusions
RBO is required for endocytosis in neuronal synapses and non-neuronal cells.
The endocytic requirement for RBO becomes more apparent in the syx3-69 background, due to increased fusion.
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Rolling Blackout: Cellular Role in Endocytosis
In rbots NMJs neuronal activity fails to trigger the formation of endosomal-like structures
a)RBO is required for direct bulk uptake of membrane into the terminal
orb) the fusion of synaptic vesicles internalized by clathrin-mediated endocytosis to form endosomal-like structures.
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Future directions
• Interaction with syx3-69 due to increased fusion. Interaction with “open” conformation syntaxin?
TMHA/H1 HB/H2 HC
28 62 71 104 111 144
SNAREH3
185 266 288
NH2
0
Linker
258
T254I
COOH
L168, and E169
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Future Directions
Characterize the endosomal-like compartments
Is RBO required for other forms of endocytosis?
Genetic screens to identify enhancers/suppressors of TS paralysis
Protein interactors of RBO
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Future Directions
Genetic interaction between rbots and shibirets1 Synthetic lethality
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Future Directions
Synergism with weaker shibire alleles?
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Model: Lipase or Scaffolding Protein?
RBO acts as a lipase to modify lipid levels and is directly responsible to PIP and PIP2 changes previously reported
Baird et al., JCB 2008
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AcknowledgementsAdvisor: Kendal Broadie, Ph.DBroadie Lab
Fu-De Huang, Ph.DHeinrich Matthies, Ph.DRalf Mohrman, Ph.D
Elvin Woodruff IIIJeffrey Rohrbough, Ph.DCheryl, Gatto, Ph.DScott Phillips, PhDCharles Tessier, Ph.DGracie Andrews, Ph.DSarah Yang, Ph.DEmma RushtonAshleigh LongLane CoffeeNeil DaniBrad RobinsonQing-xia ChenNicole Bibus-Christianson
CollaboratorsJohn McLean, Ph.D, Dept of ChemistryMichal KlimanLily Wang, Ph.D, Dept of biostatisticsJon Tapp, Vanderbilt Kennedy CenterJohn York, Ph.D, (Duke)Jessica Monserrate, Ph.D (Duke)
Dissertation Committee:Roger Colbran, Ph.D (Chair)Randy Blakely, Ph.DTodd Graham, Ph.DKendal Broadie, Ph.D (Advisor)
Neuroscience ProgramElaine Sanders-Bush, Ph.DLou Defelice, Ph.DMark Wallace, Ph.DDouglas McMahon, Ph.DMary, Early-Zald, Ph.DMary Michal-Woolman, Shirin PulousRoz Johnson
Funding: NIH grants NS41740 andGM54544