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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
Why bother with Drosophila?
http://www.ipmc.cnrs.fr/~duprat/neurophysiology/brain.htm
http://www.sciencemag.org/sciext/vis2005/show/images/slide1_large.jpg
Forward Genetics
Mutagenize
Screen for conditional temperature-sensitive (TS)
paralysis
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?
Temperature-sensitive paralysis in rbots
Flies @37OC: Selected time points from 0-6 minutes of heat shock
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
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
rbots: Neurotransmission Defect
rbots WT
Dorsal longitudinal flight muscle (DLM)
Stimulate
Record
Interneuron
Huang et al., The Journal of Neuroscience, March 1, 2006
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
Genetic interaction with syntaxints
Richmond and Broadie, Curr Opin Neurobiol 2002 , 12, 499-507
Huang et al., The Journal of Neuroscience, March 1, 2006
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
Glycine to Aspartic acid
Temperature–sensitive site
GXSXG
Aspartate/Glutamate
RBO a lipase?
H S G-D D0 289 358 527 719
G-X- -X-G
834
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.
xProposed Model for RBO Function
1) Activity –dependent loading
Exocytosis Endocytosis
Assay for imaging synaptic vesicle cycling: FM dyes
FM1-43
2) Destaining of loaded dye-assay for exocytosis
Assay for imaging synaptic vesicle cycling: FM dyes
EndocytosisExocytosis
FM1-43
Drosophila 3rd instar larval neuromuscular junction (NMJ)
7 6 13 12
4
8
5
a
p
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
FM1-43 Dye Loading Defects in rbots
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
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
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
RBOEGFP Expression in Cultured Pupal Neurons
RBO localizes to functional synapses with cycling synaptic vesicles
RBO facilitates endocytosis in central brain synapses
rbo-EGFP
rbo mutants show defects in tracer uptake in Garland cells
Garland cell UltrastructureB
C
37o C
37o C
OR rbots
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
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
Ultrastructure: rbo mutants show defects in FM1-43 uptake into Endosomes/cisternae at the NMJ
OR rbo
Ultrastructure: rbo mutants show defects in FM1-43 uptake into Endosomes/cisternae at the NMJ
Why does the loss of function syx3-69 mutation exacerbate the rbots phenotype at the NMJ?
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
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.
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.
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
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
Future Directions
Genetic interaction between rbots and shibirets1 Synthetic lethality
Future Directions
Synergism with weaker shibire alleles?
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
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