Past Department Affiliation: Post-Doctoral Fellow in Cell Biology, JHMI until Dec'02
Present Departmental Affiliation: Assitant Professor , Biology Department , The Catholic University of America
Education: Ph.D Cell, Molecular and Structural Biology from Northwestern University Medical School; B.S. Cell Biology and B.A. Communication Studies from the University of Kansas.
Telephone Number: 202-319-6681
Email Address: firstname.lastname@example.org
is the major phosphoinositide 3-kinase regulator of apical membrane
dynamics in polarized cells.
Regulation of the cellular itineraries of apical plasma membrane (PM) proteins in polarized hepatocytes is complex and poorly understood. We examined the possible role(s) of phosphoinositide 3-kinases by microinjecting inhibitory antibodies and proteins into WIF-B cells. We found that Vps34p (whose sole substrate is phosphatidylinositol) regulates endocytic trafficking of apical PM proteins from the apical surface and when inhibited, recapitulated the defects observed in wortmannin-treated cells. Injection of anti-Vps34p antibodies induced the formation of large vacuoles that contained lysosomal membrane proteins and apical proteins internalized from the apical PM. Ultrastructural analysis of the vacuoles revealed small intralumenal vesicles. Antibody-antigen trafficking assays revealed that both Vps34p and p85/p110? mediate basolateral to apical transcytosis, but at different transport steps. To further understand how Vps34p was functioning, we examined its membrane association and that of the PI3(P)-binding proteins, EEA1 and HRS, in control and wortmannin-treated cells. In control cells, 95% of Vps34p, 50% of HRS and 25% of EEA1 fractionated with membranes. HRS and Vps34p remained membrane-associated during treatment while EEA1 dissociated within 30 min. Morphologically, EEA1 distributed to puncta at the cell periphery and near the apical PM. No colocalization with early endosomal proteins was observed, but the sub-apical structures contained M6P-R. In wortmannin-treated cells, EEA1 remained membrane-associated and localized to tubules emanating from the sub-apical puncta. After longer treatment, EEA1 associated with a subpopulation of vacuoles that contained apical proteins. Similar vacuolar staining was observed in anti-Vps34p-injected cells. EEA1 and another putative binding partner, syntaxin 13, did not colocalize in control cells, but in wortmannin-treated cells, syntaxin 13 associated with the vacuoles. Together, these results indicate that Vps34p regulates hepatic apical membrane dynamics through a complex mechanism that may differ from that described for nonpolarized cells.
Apical plasma membrane proteins are sorted to a novel recycling compartment in nonpolarized hepatic and kidney-derived epithelial cells.
P.L. Tuma, L.K. Nyasae and A.L. Hubbard
The establishment and maintenance of cell surface polarity is imperative for proper epithelial cell function, but the mechanisms that regulate these processes are poorly understood. We have been investigating the molecular basis of apical plasma membrane (PM) biogenesis in polarized epithelial cells by examining the intracellular itineraries of resident apical PM proteins. To determine whether the mechanisms regulating PM protein dynamics are distinct in polarized cells, we have examined trafficking of GPI-anchored, type 1 and type 2 apical PM proteins in three classes of hepatic cells. The first class consists of polarized and differentiated cells (WIF-B), the second includes nonpolarized and differentiated cells (Fao) and the third is nonpolarized and nondifferentiated cells (Clone 9). An analogous set of kidney-derived cells was also examined: fully polarized MDCK cells, low-density, nonpolarized MDCK cells and NRK cells. In polarized cells, the apical PM proteins were expressed exclusively at the apical domain whereas in both classes of nonpolarized cells, the apical PM proteins were expressed at the cell surface as expected, but also in an intracellular compartment. Double labeling of Fao apical proteins with organelle markers for the biosynthetic pathway, lysosomes, early, late, or recycling endosomes indicated that the compartment is novel. By monitoring and quantifying the trafficking of antibody-labeled molecules in the nonpolarized cells, we determined that apical proteins were rapidly internalized and transported to the intracellular compartment and more rapidly recycled back to the PM. However, basolateral PM proteins and transferrin receptor were excluded from these pathways indicating that selective sorting to this compartment exists. These results suggest that polarized trafficking patterns are present in nonpolarized cells.