Rolling Blackout (RBO), a Drosophila EFR3 homolog, is an integral membrane protein predicted to be a lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis following a 25-37oC temperature shift, an impairment previously attributed to blocked synaptic vesicle exocytosis. rbots displays a synergistic interaction with a dominant positive Syntaxin-1A TS allele, syx3-69. At neuromuscular synapses, rbots shows a defect in styryl FM dye endocytosis, and rbots;syx3-69 double mutants display a synergistic, more severe endocytosis impairment. rbots synapses in primary brain culture show defective FM endocytosis. Nephrocyte Garland cells show the same endocytosis defect in tracer uptake assays. Ultrastructurally, rbots displays a defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there is total blockade of endosome formation possibly via activity-dependent bulk endocytosis. A point mutation in the putative lipase active site fails to rescue embryonic lethality of the null mutants and garland cell endocytosis defects in the TS background, suggesting that the mutation disrupts some vital aspect of protein function and that lipase activity may be required for membrane trafficking and viability. However, the putative lipase-dead mutant is able to fully rescue paralysis and NMJ endocytic defects in rbots possibly indicative of interallelic complementation. Further, we identified a synthetic lethal interaction between rbots and the dynamin GTPase mutant shits1. In all assays, both in non-neuronal cells and neuronal synapses, shits1; rbots phenocopies shits1 endocytic defects, indicating that dynamin and RBO act in the same pathway. Together, these results demonstrate that RBO is required for dynamin-dependent bulk endocytosis/macropinocytosis in both neuronal and non-neuronal cells, and at the synapse this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03292010-133228 |
| Date | 29 March 2010 |
| Creators | Vijayakrishnan, Niranjana |
| Contributors | kendal broadie, randy blakely, todd graham, roger colbran |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu//available/etd-03292010-133228/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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