thesis research

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