Phosphatidylinositol 4,5 bisphosphate (PI(4,5)P<sub>2</sub>) plays a crucial role in cellular signalling. Functions include the generation of the key second messengers, inositol 1, 4, 5 triphosphate and diacylglycerol, as well as direct regulation of the activity of proteins that mediate cytoskeletal attachment, vesicular transport, ion-channel activation and enzyme activation. Despite multiple cellular functions, the total level of PI(4,5)P<sub>2</sub> shows minimal changes. This thesis aims at investigating the mechanisms by which the PI(4,5)P<sub>2</sub> levels are regulated in <i>Drosophila </i>photoreceptors. Two distinct aspects of PI(4,5)P<sub>2</sub> signalling were addressed. (i) Microvillar membrane extracts were used for <i>in vitro </i>binding assays to identify PI(4,5)P<sub>2</sub> binding proteins. The results define an experimental approach by which the identity of PI(4,5)P<sub>2</sub> binding proteins in photoreceptors can be revealed. (ii) We also studied the principal route by which PI(4,5)P<sub>2</sub> might be synthesised in <i>Drosophila </i>photoreceptors. To test the hypothesis that CG3682, the <i>Drosophila </i>putative Type I PI4P 5-kinase, performs its function in <i>Drosophila </i>photoreceptors, we generated a loss-of-function mutant of CG3682 by homologous recombination. Analysis by mass spectrometry showed that the pool of phosphatidylinositol monophosphates (PIP) was elevated in the mutant retinae, suggesting that this gene product regulates the conversion of PI4P to PI(4,5)P<sub>2</sub>. This mutant showed a profound defect in the light response with a ca. 500 fold reduction in sensitivity to light compared to the wild type but minimal defects in photoreceptor ultrastructure. These findings together suggest that CG3682 contributes to the Type I PIPkin activity that underpins PI(4,5)P<sub>2</sub> synthesis during phototransduction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:597392 |
| Date | January 2010 |
| Creators | Chakrabarti, P. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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