Biomedical Sciences Graduate Program(s)
Biomedical Sciences Graduate Programs
Arf-family GTPases; molecular switches controlling vesicular transport and cytoskeleton assembly: The Arfs are a family of six small, Ras-like GTP-binding proteins that are important regulators of vesicular transport in all eukaryotic cells. Generally speaking, the Arfs nucleate the assembly of coat protein complexes at sites of carrier vesicle formation, and it is these coats that both select cargo for transport and deform the donor membrane to form a vesicle. We are particularly interested in the function of Arfs in endocytosis and the post-endocytic sorting and trafficking of membrane proteins. We are using two complementary approaches to define the function of individual Arfs in these processes; RNAi-mediated knockdowns and live imaging of cells expressing fluorescently tagged Arf isoforms. Our studies indicate that at least three Arfs act on endosomal membranes and we are currently working to define the adaptor molecules recruited by each Arf and how specific cargo molecules are partitioned among them for transport.
Arf function in the brain: The human genome encodes 15 guanine nucleotide exchange factors (GEFs) that activate Arfs by displacing bound GDP and facilitate loading with GTP. A subset of these, the BRAGs (Brefeldin-resistant Arf GEFs) are highly enriched in the brain, where they localize to postsynaptic densities. Recently, mutations in BRAG1 were identified in families with heritable X-linked mental disability, indicating an important role for BRAGs in synaptic transmission. Using a combination of biochemical, cell biological and electrophysiological approaches, we recently found that BRAG1 controls the strength of synaptic responses by modulating the number of neurotransmitter (AMPA) receptors in the synaptic membrane. Mutations in the catalytic domain and a calmodulin-binding motif that mimic those reported in patients with mental disability have distinct and dissociable effects on neurotransmission, providing a mechanistic explanation for the observed cognitive deficits in these patients. The BRAGs are large proteins with multiple protein-protein interaction modules, and we are currently working to define the specific binding partners associated with each isoform, and how they coordinate BRAG function with other signaling events in the synapse.
Cell-based studies of Salmonella infection: Salmonellae penetrate the intestinal epithelium by injecting an array of effector proteins into the cytoplasm of epithelial cells that trigger phagocytic uptake of attached bacteria. We are interested in defining the cellular targets of these effector proteins, and how their manipulation by the bacteria promotes their internalization and intracellular survival. Using yeast two-hybrid screens and shRNA screens, we have identified a number of host proteins that either interact directly with bacterial effectors or are necessary for bacterial uptake or intracellular proliferation. We recently reported that the tip of the bacterial translocation apparatus (Type III secretion system), a protein called SipC, interacts with components of the host vesicular transport machinery (the Exocyst) and directs secretory vesicles to sites of bacterial attachment, where the membrane is used to build the phagocytic apparatus. We are currently looking for graduate students/postdocs to further analyze the ³hits² from these screens and determine their function in Salmonella pathogenesis.
Mouse models: We recently found that the tyrosine kinase FAK (Focal Adhesion Kinase) is necessary for Salmonella entry into host cells. To examine the role of FAK in an in vivo model of Salmonella infection, we (in collaboration with Amy Bouton¹s lab) generated mouse lines conditionally lacking FAK in either the intestinal epithelium (villin-Cre) or cells of the macrophage/monocyte lineage (LysM-Cre). Surprisingly, we found that loss of FAK from the epithelium did not affect Salmonella colonization of orally infected mice, however mice lacking FAK in monocytes/macrophages are significantly less susceptible to infection. We are currently working to define the physiological basis of this observation, using a combination of immunohistochemistry, flow cytometry and confocal microscopy.
Innate immunity to infection: The innate immune system is the first line of defense against invading microorganisms. Phagocytes such as macrophages and neutrophils express so-called Pattern Recognition Receptors that recognize conserved motifs in bacterial/viral products. Such receptors can stimulate microbe engulfment, initiate an inflammatory response, or both. We have identified a novel receptor, BAI1, that selectively recognizes a surface component of Gram-negative bacteria, and mediates their engulfment by macrophages. Our findings suggest that BAI1 also cooperates with Toll-like receptors (specifically TLR4) to mount an inflammatory response to bacterial infection. We are currently looking for graduate students/postdocs to help characterize the interaction between BAI1 and bacteria, and define the signaling pathways used by BAI1 to trigger the inflammatory response.
Das, S., Owen, K.A., Ly, K.T., Park, D., Black, S.G., Wilson, J.M., Sifri, C.D., Ravichandran, K.S., Ernst, P.B., and Casanova, J.E. (2011). Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria. Proc Natl Acad Sci U S A 108, 2136-2141.
Nichols, CD, Casanova, JE. Salmonella-directed recruitment of new membrane to invasion foci via the host exocyst complex. Current Biology 20, 1316-1320.
Ly KT, Casanova JE. Abelson tyrosine kinase facilitates Salmonella enterica serovar Typhimurium entry into epithelial cells. Infect Immun. 2009 77(1):60-9.
Myers, KR and Casanova, JE. Regulation of actin cytoskeleton dynamics by Arf family GTPases. Trends in Cell Biology 2008 Apr;18(4):184-92.
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