Polarized epithelial cell function relies on the proper sorting and distribution of newly synthesized proteins to either the cells apical or basolateral domains. If trafficking is altered by disruptions either the fidelity or efficiency of this sorting then disease can result. There is an increasing appreciation for the role of phosphatidylinositol metabolism in membrane traffic, including the sorting and delivery of newly synthesized proteins. I have studied how biosynthetic delivery pathways are regulated by the expression of phosphatidylinositol-metabolizing enzymes. The phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme, murine phosphatidylinositol 4-phosphate 5-kinase I alpha (PI5KIá) localizes to the apical pole of Madin-Darby canine kidney (MDCK) cells and increases cellular PIP2 concentrations over control cell levels. Interestingly, expression of exogenous PI5KIá stimulated the rate of surface delivery of a subset apical proteins that associate with lipid rafts, including influenza hemagglutinin (HA). Conversely, overexpression of the PIP2-5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe), which is defective or absent in patients with Lowe syndrome, decreased cellular PIP2 levels and inhibited the rate of HA delivery.
The observation that increases in PIP2 stimulate apical delivery of HA suggests the possibility that depletion of OCRL may have a similar effect to overexpression of PI5K. I used siRNA to knock down OCRL in MDCK cells and human proximal tubule (HK2) cells and examined the consequence on HA surface delivery. Knockdown of OCRL slightly increased cellular PIP2 levels but did not stimulate HA delivery.
PI5K-mediated increases in PIP2 results in activation of neuronal Wiskott-Aldrich syndrome protein (N-WASP) leading to downstream actin cytoskeleton rearrangements including the formation of actin comets. To examine the potential role of N-WASP in HA delivery I expressed a dominant negative inhibitor of N-WASP function, the WA domain from a WASP family member, WAVE1. Expression of the WA domain significantly and selectively inhibited the rate of HA surface delivery. siRNA-mediated knockdown of N-WASP also inhibited HA delivery, confirming a role for N-WASP in biosynthetic traffic. Consistent with this, PI5KIá and HA (but not p75) were visualized on actin comets in formaldehyde-fixed MDCK cells. In summary, my data support a role for PI5K-stimulated actin comet formation in apical delivery of a subset of newly synthesized proteins.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-10142008-141102 |
| Date | 14 October 2008 |
| Creators | Guerriero, Christopher James |
| Contributors | Meir Aridor, Ora A. Weisz, Gerard L. Apodaca, Jes K. Klarlund, Paul R. Kinchington |
| 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-10142008-141102/ |
| 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. |
Page generated in 0.0015 seconds