| Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain, and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cell polarity and membrane trafficking in several cell types. In this dissertation, I examined whether this protein regulates tight junction function in Madin-Derby canine kidney (MDCK) cells and the pathways that direct proteins to the apical and basolateral surfaces of these cells. I found that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, while expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis, but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.; Upon the establishment of epithelial cell architecture, vesicular trafficking along the endocytic pathway enables these cells to perform two important functions; the vectorial transport of ions and solutes and the detection/response to stimuli from the extracellular milieu. RhoB, another member of the Rho family, has been localized to several cellular sites within nonpolarized cells including endosomes and Golgi, however, its cellular distribution and function in the endocytic pathway of epithelial cells remains unknown. Using confocal microscopy, I found that endogenous RhoB and green fluorescent protein tagged-wild-type RhoB were localized to early endosomes, and to a much lesser extent to late endosomes or Golgi of fixed and live MDCK cells. Consistent with its localization to endosomes, I observed that expression of RhoBV14 inhibited basolateral recycling of transferrin, degradation of epidermal growth factor, and apical recycling of IgA. While expression of RhoBV14 inhibited transcytosis of IgA, expression of RhoBN19 stimulated IgA transcytosis by modulating transit between basolateral early endosomes and more apical endosomes. My results indicate that RhoB is localized, in part, to early endosomes where it appears to regulate transit of receptors through the early endocytic system. |
