Phosphatidylinositol (PI) and its phosphorylated derivatives, phosphatidylinositides (PIPs), are versatile cellular regulators participating in myriad events including signal transduction, cytoskeleton organization, protein targeting and many steps of membrane traffic. Different PIPs exhibit non-overlapping distributions on cellular membranes. This feature contributes to organelle identities and is tightly controlled by kinase/phosphatase-mediated PIP synthesis and turnover. Mechanisms regarding compartment-restriction and detailed functions of many PIPs and PI/PIP metabolizing enzymes remain largely unknown. My dissertation focuses on the cellular targeting mechanism of a PIP kinase and the pathogenesis of a disease caused by mutations in a PIP phosphatase.
Phosphatidylinositol (4,5)-bisphosphate (PIP2), an apical-surface-enriched PIP in polarized epithelial cells, is primarily synthesized via phosphorylation of phosphatidylinositol 4-phosphate (PI4P) in the presence of type I PI 5-kinases (PI5KIs). Previous studies have suggested that the three isoforms of PI5KI (¦Á, ¦Â, and ¦Ã) exhibit distinct cellular functions. Data from our lab indicate that these three PI5KIs are differentially localized in polarized renal cells. While the majority of ¦Á and ¦Ã isoforms are present on lateral cell surface, the ¦Â isoform strikingly localizes to the apical plasma membrane. Using mutagenesis, immunofluorescence, and confocal microscopy, I have found that the apical surface distribution of PI5KI¦Â is nonsaturable and does not require catalytic activity or the presence of PIP2. These results provide useful information for future studies on PI5KI¦Â-regulated cellular activities.
PIP2 turnover can be catalyzed by a variety of enzymes, one of which is OCRL1. OCRL1 is a PI 5-phosphatase that preferentially hydrolyzes PIP2, producing PI4P, and is associated with the trans-Golgi network, endosomes, and clathrin-coated-pits. Genetic defects of OCRL1 cause Lowe syndrome, a disease manifested by congenital cataracts, mental retardation, and renal tubular dysfunction. By examining cultured renal epithelial cells acutely depleted of OCRL1 via RNA interference, I have found that loss of OCRL1 does not interfere with endocytic trafficking of the multiligand receptor megalin, or uptake of megalin ligands. OCRL1 knockdown did appear to disrupt delivery of newly-synthesized lysosomal hydrolases and alter distribution of primary cilia length in renal epithelial cells. These findings suggest that multiple pathways may contribute to development of renal symptoms in Lowe patients.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12192010-002915 |
| Date | 20 December 2010 |
| Creators | Cui, Shanshan |
| Contributors | Ora A. Weisz, Neil A. Hukriede, Linton M. Traub, Rebecca P. Hughey, Carolyn B. Coyne |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12192010-002915/ |
| Rights | restricted, 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 University of Pittsburgh 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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