James E. Casanova, PhD
Graduate School: Wesleyan University
Primary Appointment: Professor, Cell Biology
Role of Arf family GTPases in vesicular transport and cytoskeleton assembly.
Cell Biology of bacterial pathogenesis.
The innate immune response to bacterial infection.
Email Address: email@example.com
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
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.
Moravec R, Conger KK, D'Souza R, Allison AB, Casanova JE. BRAG2/GEP100/IQSec1 interacts with clathrin and regulates α5β1 integrin endocytosis through activation of ADP ribosylation factor 5 (Arf5). J Biol Chem. 2012 Sep 7;287(37):31138-47. doi: 10.1074/jbc.M112.383117. Epub 2012 Jul 19.
Myers KR, Wang G, Sheng Y, Conger KK, Casanova JE, Zhu JJ. Arf6-GEF BRAG1 regulates JNK-mediated synaptic removal of GluA1-containing AMPA receptors: a new mechanism for nonsyndromic X-linked mental disorder. J Neurosci. 2012 Aug 22;32(34):11716-26. doi: 10.1523/JNEUROSCI.1942-12.2012.
Casanova JE. Advantages and limitations of cell-based assays for GTPase activation and regulation. Cell Logist. 2012 Jul 1;2(3):147-150.
Das S, Owen KA, Ly KT, Park D, Black SG, Wilson JM, Sifri CD, Ravichandran KS, Ernst PB, Casanova JE. 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. 2011 Feb 1;108(5):2136-41. doi: 10.1073/pnas.1014775108. Epub 2011 Jan 18.
Owen KA, Abshire MY, Tilghman RW, Casanova JE, Bouton AH. FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing. PLoS One. 2011;6(8):e23123. doi: 10.1371/journal.pone.0023123. Epub 2011 Aug 24.
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