Rolling blackout is required for synaptic vesicle exocytosis.
Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate-diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.
Huang
Fu-De
FD
Department of Biological Sciences, Kennedy Center for Research on Human Development, Brain Institute, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
Woodruff
Elvin
E
Mohrmann
Ralf
R
Broadie
Kendal
K
D000818:Animals; D030801:Animals, Genetically Modified; D001522:Behavior, Animal; D015153:Blotting, Western / Q000379:methods; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000502:physiology*; D002352:Carrier Proteins; D003952:Diagnostic Imaging / Q000379:methods; D004307:Dose-Response Relationship, Radiation; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D004558:Electric Stimulation / Q000379:methods; D019706:Excitatory Postsynaptic Potentials / Q000235:genetics / Q000528:radiation effects; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D006735:Horseradish Peroxidase / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007814:Larva; D008297:Male; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008959:Models, Neurological; D009068:Movement / Q000502:physiology; D009154:Mutation / Q000502:physiology; D009412:Nerve Fibers / Q000502:physiology / Q000528:radiation effects; D009469:Neuromuscular Junction / Q000235:genetics / Q000502:physiology / Q000528:radiation effects / Q000648:ultrastructure; D011930:Reaction Time / Q000502:physiology / Q000528:radiation effects; D050600:SNARE Proteins / Q000378:metabolism; D009435:Synaptic Transmission / Q000235:genetics / Q000502:physiology*; D013572:Synaptic Vesicles / Q000502:physiology* / Q000648:ultrastructure; D013697:Thermosensing / Q000235:genetics / Q000502:physiology*; D013997:Time Factors
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D029721:Drosophila Proteins; D050600:SNARE Proteins; C083141:phorbol ester binding protein; D006735:Horseradish Peroxidase; D002265:Carboxylic Ester Hydrolases; C494132:stmA protein, Drosophila