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Title: Cellular roles for proteins linked to phosphatidylinositol (3,5)-bisphosphate metabolism
Author: Whittingham, Jane Louise
ISNI:       0000 0004 2671 2743
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2008
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The phosphoinositide lipids Ptdlns(3)P and Ptdlns(3,5)P2 play important roles on the endocytic pathway. Ptdlns(3)P localises to early endosomes and multivesicular bodies (MVBs) and is proposed to recruit proteins involved in fusion of early endosomes and internalisation of ubiquitinated receptors. Ptdlns(3,5)P2 is proposed to be involved in terminal maturation of Iysosomes and endosome to Golgi transport, but the precise role of this lipid in mammalian cells remains unclear. Studies in yeast have identified various proteins associated with Ptdlns(3,5)P2 metabolism, for which mammalian homologues have been found. These are the Ptdlns(3)P5- kinase Fab1/PIKfyve, Fig4 a 5-phosphatase that dephosphorylates Ptdlns(3,5)P2, Vac14 which has been shown to act as an upstream activator of PIKfyve, and WIPI-2/Svp1 a putative downstream effector of Ptdlns(3,5)P2. In this study I have further characterised three of these proteins and examined the cellular roles of all four with respect to a variety of trafficking pathways in which Ptdlns(3,5)P2 has been implicated. siRNA was used to examine the knockdown phenotypes of each of these proteins. Furthermore, I directly compared for the first time the effects of knockdown of PIKfyve, with acute pharmacological inhibition of its enzymatic activity. Loss of PIKfyve activity caused a failure in the retrieval of a variety of different cargos to the trans-Golgi network (TGN), including mannose-6-phosphate receptors, responsible for efficient delivery of lysosomal enzymes, and the TGN resident protein TGN46, leading to their accumulation in dispersed punctae. A failure in tyrosine kinase receptor downregulation was also observed following combined knockdown of PIKfyve with either Vac14 or Fig4 or following pharmacological inhibition of PIKfyve. PIKfyve knockdown alone had no effect, suggesting a low threshold of Ptdlns(3,5)P2 is necessary and sufficient for this pathway. Svp1 is the best characterised Ptdlns(3,5)P2 effector in yeast and is also an autophagy-related gene (Atg18), thereby implicating Ptdlns(3,5)P2 in this process. A family of putative mammalian Svp1 homologues have been identified, known as the WIPI family. I investigated the role of Ptdlns(3,5)P2 and WIPI-2 in mammalian autophagy. By monitoring formation of the autophagosome marker GFP-LC3 II, PIKfyve and WIPI-2 were found to have opposite effects. Furthermore, WIPI-2 redistributes to punctate structures upon induction of autophagy, which partially colocalise with autophagosome markers, in a manner dependent not on PIKfyve but on PI(3)-kinase activity. The evidence presented suggests that Ptdlns(3,5)P2 may playa role in mediating the maturation of a subset of MVBs, leading to swelling of endosomal compartments and rendering the MVBllate endosome or autophagosomes refractory to fusion with the lysosome in cells depleted of PIKfyve.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral