1. Cellular Role of the Drosophila EFR3 Homolog Rolling Blackout (RBO) in Synaptic Transmission NiranjanaVijayakrishnan Dissertation Defense, March 10th, 2010 Broadie Lab

2. Why bother with Drosophila? http://www.sciencemag.org/sciext/vis2005/show/images/slide1_large.jpg http://www.ipmc.cnrs.fr/~duprat/neurophysiology/brain.htm

3. Forward Genetics Mutagenize Screen for conditional temperature-sensitive (TS) paralysis

4. Synaptic transmission Where does Rolling Blackout (RBO) function? paralytic : A subunit of Na+ channel Exocytosis syx3-69 Voltage Gated Na+ channel Endocytosis shibire, GTPasedynamin Action Potential, initiation, propagation

5. Temperature-sensitive paralysis in rbots Flies @37OC: Selected time points from 0-6 minutes of heat shock

7. G527D missense mutation causes TS phenotype

8. Homologs from yeast to humans

10. rbots: Neurotransmission Defect Record Dorsal longitudinal flight muscle (DLM) Stimulate Interneuron rbots WT Huang et al., The Journal of Neuroscience, March 1, 2006

11. rbots:SynapseUltrastructure rbots at 37 C: docked vesicles 22 C 37 C WT rbo # of docked vesicles per active zone rbo Huang et al., The Journal of Neuroscience, March 1, 2006

12. Genetic interaction with syntaxints Richmond and Broadie, Curr Opin Neurobiol 2002 , 12, 499-507 Huang et al., The Journal of Neuroscience, March 1, 2006

13. Na+ and Ca2+ Ga ?? b g Drosophila Vision Cascade Complete loss of light-dependent receptor potential Light TRP PIP2 DAG+ IP3 G protein coupled Receptor PLC PLC= Phospholipase C b DAG=Diacylglycerol PIP2= Phosphotidylinositiol (4,5) bis phosphate Garcia-Murillas et al., Neuron 49, 533–546, February 16, 2006 TRP= Transient Receptor Potential

14. Temperature–sensitive site Aspartate/Glutamate GXSXG Glycine to Aspartic acid

15. RBO a lipase? 0 289 358 527 719 H S G-D D G-X- -X-G 834

16. rolling blackoutts (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 temperature Ultrastructure: Vesicle accumulation in DLM boutons Increase in vesicles at active zone (docked vesicles) Synergistic genetic interaction with t-SNARE Syntaxin 1A-rbots; syxtsmutants paralyze at 33oC Homology to known lipases HPTLC analysis of lipids revealed an increase in overall PIP and PI(4,5)P2 levels and a decrease in DAG levels.

17. x Proposed Model for RBO Function

18. Assay for imaging synaptic vesicle cycling: FM dyes Exocytosis Endocytosis FM1-43 1) Activity –dependent loading

19. Assay for imaging synaptic vesicle cycling: FM dyes Endocytosis Exocytosis FM1-43 2) Destaining of loaded dye-assay for exocytosis

20. Drosophila 3rdinstar larval neuromuscular junction (NMJ) a p 7 6 13 12 4 5 8

21. Depolarization-dependent FM1-43 Dye Loading: Assay Vesicle Exocytosis O O 25 C Load 37 C Unload Protocol 1 OR O 37 C syx syx O O 25 C 25 C rbo rbo rbo;syx + rbo;syx 60 mM K Imaging FM1-43

22. FM1-43 Dye Loading Defects in rbots

23. Depolarization-dependent FM1-43 Dye Loading: Assay Vesicle Endocytosis O O 37 C Load 25 C Unload OR syx Protocol 2 rbo O 37 C rbo;syx + 60 mM K O O Imaging 25 C 25 C FM1-43

24. FM1-43 Dye Loading Defects in rbots p<.001 p<.001 25 oC p<.001 37 oC Mean Fluorescence Intensity L L L L L L L UL UL UL UL UL UL UL UL L OR OR syx rbo rbo rbo;syx rbo;syx syx Protocol 2 Protocol 1

25. Endocytic defects in rbots mutants are rescued by the wild-type rbo gene O 37 C O O 25 C 25 C wt rbo-eGFP in rbots/D rbo Rescue Fluorescence Intensity (AU) OR rbo Rescue

26. RBOEGFP Expression in Cultured Pupal Neurons

27. RBO localizes to functional synapses with cycling synaptic vesicles

28. RBO facilitates endocytosis in central brain synapses

29. rbo mutants show defects in tracer uptake in Garland cells rbo-EGFP

30. Garland cell Ultrastructure B rbots OR 37oC C 37oC

31. Ultrastructure: rbo mutants show defects in tracer uptake in Garland cells rbo OR Number of Endosomes/section Block in Horse RaddishPeroxidase uptake into endosome at 37oC in rbo

32. Ultrastructure: rbo mutants show defects in Endosome/cisternae formation at the NMJ OR rbots OR rbo 37oC Cisternae Number 37oC 10 min Hi [K+] Rest Stim

33. Ultrastructure: rbomutants show defects in FM1-43 uptake into Endosomes/cisternae at the NMJ OR rbo

34. Ultrastructure: rbomutants show defects in FM1-43 uptake into Endosomes/cisternae at the NMJ

35. Why does the loss of function syx3-69 mutation exacerbate the rbots phenotype at the NMJ?

37. At 25oC: increase in “mini” frequency and amplitude of evoked release

39. 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 or b) the fusion of synaptic vesicles internalized by clathrin-mediated endocytosis to form endosomal-like structures.

40. Future directions Interaction with syx3-69 due to increased fusion. Interaction with “open” conformation syntaxin? L168, and E169 28 62 71 104 111 144 266 288 0 258 185 TM HA/H1 HB/H2 HC SNARE H3 COOH Linker NH2 T254I

41. 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

42. Future Directions Genetic interaction between rbots and shibirets1 Synthetic lethality

43. Future Directions Synergism with weaker shibire alleles?

44. 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

45. Acknowledgements Broadie Lab Collaborators John McLean, Ph.D, Dept of Chemistry Michal Kliman Lily Wang, Ph.D, Dept of biostatistics Jon Tapp, Vanderbilt Kennedy Center John York, Ph.D, (Duke) Jessica Monserrate, Ph.D (Duke) Advisor: Kendal Broadie, Ph.D Elvin Woodruff III Jeffrey Rohrbough, Ph.D Cheryl, Gatto, Ph.D Scott Phillips, PhD Charles Tessier, Ph.D Gracie Andrews, Ph.D Sarah Yang, Ph.D Emma Rushton Ashleigh Long Lane Coffee Neil Dani Brad Robinson Qing-xia Chen Nicole Bibus-Christianson Dissertation Committee: Roger Colbran, Ph.D (Chair) Randy Blakely, Ph.D Todd Graham, Ph.D Kendal Broadie, Ph.D (Advisor) Neuroscience Program Elaine Sanders-Bush, Ph.D Lou Defelice, Ph.D Mark Wallace, Ph.D Douglas McMahon, Ph.D Mary, Early-Zald, Ph.D Mary Michal-Woolman, ShirinPulous Roz Johnson Fu-De Huang, Ph.D Heinrich Matthies, Ph.D Ralf Mohrman, Ph.D Funding: NIH grants NS41740 and GM54544