The endocytic and postendocytic trafficking pathways in polarized epithelial cells was examined. First, sorting of fluid and membrane internalized from the apical plasma membrane was analayzed. Internalized fluid and membrane were intially delivered to apical early endosomes (AEE). Fluid remained in the AEE while membrane was rapidly sorted and delivered to the Rab11+ apical recycling endosome (ARE). The delivery of membrane markers to the ARE was microtubule-dependent. Transferrin, a marker of basolateral recycling pathway, had access to the AEE but not the ARE. Next, the role of Rac1 and RhoA in endocytosis and postendocytic was determined. Both Rac1 and RhoA were involved in regulation of endocytosis from both plasma membrane domains. Furthermore, Rac1 was implicated in regulation of apically targeted traffic from both the endocytic and secretory pathways. Expression of dominant active Rac1 (Rac1V12) caused formation of a central aggregate of membranes composed in part of the ARE. Markers targeted for the apical plasma membrane were trapped within this aggregate. RhoA was involved in the regulation of traffic from basolateral early endosomes (BEE). Expression of dominant active RhoA (RhoAV14) trapped ligands internalized from the basolateral plasma membrane in BEE that were also associated with filamentous actin (F-actin). A subset of BEE from control cells were also f-actin positive. Colocalization of BEE with proteins involved in actin polymerization based propulsion (APBP) and myosin motor-based propulsion was determined. Proteins involved in APBP were not associated with BEE bu MIc, a type I myosin, did colocalize with a subset of BEE. This suggests that BEE are transported along associated f-actin to the level of the stress fibers carried by MIc. Finally, the role of SNAP-23 in basolateral recycling of transferrin was determined. SNAP-23 was localized to the endosomes throughout the cell and to the basolateral plasma membrane. Treatment of permeabilized MDCK cells with botulinum neurotoxin E or addition of exogenous SNAP-23 or anti-SNAP-23 antibodies all inhibited transferrin recycling. This suggests that SNAP-23 is important for basolateral recycling in polarized epithelial cells.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-02282002-133438 |
| Date | 25 April 2002 |
| Creators | Leung, Som-Ming |
| Contributors | Dr. Ora Weisz, Dr. John Johnson, Dr. Rebecca Hughey, Dr. Sandra Murray |
| 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:80/ETD/available/etd-02282002-133438/ |
| 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 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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