University of Virginia Health System

Research in this laboratory is aimed at understanding the mechanisms by which membrane proteins are targeted to their appropriate cellular destinations in eukaryotic cells. Using a variety of cultured cell systems, we are studying the processes of endocytosis and, in epithelial cells, transcytosis, which is the process of transepithelial vesicular transport. For studies of epithelial transport we use a model system in which MDCK cells are cultured on permeable filter supports, which mimics closely the biology of epithelial tissues in vivo. Many aspects of membrane traffic are controlled by small monomeric GTPases of the rab and ARF (ADP-ribosylation factor) families. As described below, we are currently analyzing the function of several of these family members in both epithelia and non-polarized cells. Current research projects in the lab include: 1. Members of the ARF family of GTPases have been shown to inititate the process of vesicle formation in the Golgi apparatus (and possibly other sites) by recruiting coat proteins to the membrane. As ARFs have little intrinsic GTPase activity, they require the function of accessory proteins such as GTPase Activating Proteins (GAPs) and Guanine nucleotide Exchange Factors (GEFs). We have cloned and are characterizing an exchange factor for ARF6, which functions in the regulation of endocytosis and, perhaps integrally, with the organization of the actin cytoskeleton. The activity of this protein is regulated by inositol phospholipids, and we have recently found that it synergizes with protein kinase C to stimulate cytoskeletal reorganization. We are currently working to define the signal transduction pathways upstream and downstream of this regulatory molecule. 2. Over thirty members of the rab family of GTPases have been identified, each of which regulates a specific aspect of membrane transport. Several rabs have been found to be expressed exclusively in epithelia, and presumably regulate transport pathways that are specific to polarized cells. We have found that one of these, rab25, is localized to an apical endosomal compartment that is thought to be the primary site at which endocytosed membrane proteins are sorted for transport to other parts of the cell. We are currently attempting to determine which aspects of sorting are controlled by rab25 and its close homolog rab11, by expression of constitutively active and dominant negative mutants, using adenovirus-mediated transfection of polarized cells. 3. Salmonella invade their animal hosts by entering into and traversing intestinal epithelial cells. Invasion is initiated by inducing the host cells, which are not inherently phagocytic, to phagocytose attached bacteria, a process that involves dramatic reorganization of the apical actin cytoskeleton. After internalization, the bacteria remain enclosed in cellular membranes whose properties change as they migrate from the apical pole of the cell to the basolateral pole where the bacteria exit the cell and enter the bloodstream. We are examining the mechanisms of bacterial invasion into polarized cells, and the role of specific small GTPases in the maturation and transcytosis of the Salmonella-containing vacuole.

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