title: Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses.
abstract: Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbo(ts)) displays paralysis within minutes following a temperature shift from 25 degrees C to 37 degrees C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbo(ts) displays a strong synergistic interaction with the Syntaxin-1A TS allele syx(3-69), recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbo(ts) showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbo(ts);syx(3-69) double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbo(ts) synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbo(ts) displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbo(ts) synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.
authors.affiliation: Department of Biological Sciences, Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Niranjana
authors.initials: N
authors.lastname: Vijayakrishnan
authors.forename: Elvin A
authors.initials: EA
authors.lastname: Woodruff
authors.forename: Kendal
authors.initials: K
authors.lastname: Broadie
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000033:anatomy & histology / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D029721:Drosophila Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; C494132:stmA protein, Drosophila