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Regulation of Endocytosis and Postendocytic Traffic in Polarized Epithelial Cells

Leung, Som-Ming (2002) Regulation of Endocytosis and Postendocytic Traffic in Polarized Epithelial Cells. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Leung, Som-Mingsolst3@pitt.eduSOLST3
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairMurray, Sandra
Committee MemberJohnson, John
Committee MemberWeisz, Ora
Committee MemberHughey, Rebecca
Date: 25 April 2002
Date Type: Completion
Defense Date: 28 January 2002
Approval Date: 25 April 2002
Submission Date: 28 February 2002
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: IgA; Madin Darby Canine Kidney; Rab; SNARE; Tf; transport; vesicle
Other ID:, etd-02282002-133438
Date Deposited: 10 Nov 2011 19:31
Last Modified: 15 Nov 2016 13:36


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