Browsing by Subject "endocytosis"
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Item Characterization of the entry mechanisms utilized by the alphavirus venezuelan equine encephalitis virus to infect mosquito cells(2007-08-13) Tonya Michelle Colpitts; Robert Davey, PhD; Scott Weaver, PhD; Peter Mason, PhD; Lisa Elferink, PhD; Christopher Broder, PhDVenezuelan equine encephalitis eirus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. VEEV is an enveloped, positive-strand RNA virus that is transmitted by a mosquito vector. Most research on alphavirus entry was done with the Old World alphavirus Semliki Forest virus (SFV) in mammalian cells. Not much is known about the entry of New World alphaviruses, especially in cells of the viral vector, the mosquito. Work with SFV has shown that Old World alphaviruses enter mammalian cells via receptor-mediated, clathrin-mediated endocytosis. This endocytic pathway utilizes several proteins in the mammalian cell, including the clathrin protein, the small GTPases known as Rab proteins and the large GTPase dynamin. These proteins have been shown to play a role in the entry of several viruses and are thought to be involved in alphavirus entry in mammalian cells. Here mosquito homologs of these proteins are identified, isolated and characterized in the mosquito cell. Rab5, Rab7 and dynamin are shown to be involved and necessary for VEEV entry and infection in mosquito cells. A novel entry assay is used to confirm that VEEV requires a low pH to enter mosquito cells. This work represents the first characterization of the involvement of mosquito endocytic pathways for infection of a New World alphavirus and sheds light on an important aspect of virus infection in an insect vector. The role of actin in VEEV internalization was also examined. Actin is known to be involved in the mammalian endocytic pathway and to act together with dynamin to coordinate endocytosis. Here mosquito actin is identified and shown to colocalize with mosquito dynamin. Both proteins also colocalize with internalized VEEV. Inhibiting actin polymerization prevents entry of the virus both by microscopic examination as well as utilizing the luciferase entry assay. This work shows that VEEV enters the mosquito cell via a pH-dependent endocytic pathway that requires functional endocytic proteins including Rab5, Rab7 and dynamin. It is also shown that F-actin must be present for VEEV to enter mosquito cells and that actin and dynamin act together during virus internalization.Item Determining the role of a small GTPase, Ral, and an endocytic factor, epsin, in Drosophila Notch signaling(2011-12) Cho, Bomsoo; Fischer, Janice AnnCell-cell communication events are crucial to determine the fate of each cell during development. Notch signaling is involved in many different contexts in determining cell fate by mediating cell-cell communication. Furthermore, regulation of the Notch transduction pathway is critical for normal cellular function, which is implicated in various diseases, including cancers. At a certain developmental time point, intrinsic or extrinsic developmental cues induce biases in ligands and Notch receptors between neighboring cells. These initial biases are further amplified by various cellular factors which eventually dictate cell fates. In Drosophila, two Notch ligands, Delta and Serrate, trigger Notch receptor activation in nearby cells by virtue of numerous regulating factors. One important question in this area is how cells become Notch signal sending or receiving cells for cell fate decisions. I show evidence about a distinct mechanism for biasing the direction of Notch signaling that depends on a small GTPase, Ral, during Drosophila photoreceptor cell development. Investigations described here indicate that Fz signaling up-regulates Ral transcription in a signal sending fate cell, the R3 precursor, and Ral represses ligand-independent activation of Notch in the R3 precursor. This event ensures R3 to become a signaler and contributes to asymmetric Notch activation in the neighboring cell, R4. Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, possible Ral effector pathways that are important for Notch regulation will be proposed. To trigger Notch activation in adjacent cells, Notch ligand endocytosis by the signaling cells is necessary. Recently, it was suggested that control of membrane trafficking is important not only for ligand signaling, but also for Notch receptor activation. Furthermore, Notch receptor trafficking regulates critical cellular functions, including proliferation, which is implicated in tumors. Therefore, another important question in Notch signaling is about the role of membrane trafficking in regulation of the Notch transduction pathway. Drosophila endocytic epsin, Liquid facets [Lqf], is a key component necessary for ligand endocytosis, thereby triggering Notch activation in adjacent cells. However, its function in signal receiving cells for Notch activation has not been studied. In this dissertation, I provide evidence that epsin is also required in signal receiving cells for Notch activation in developmental contexts. Furthermore, genetic and molecular evidence suggests that epsin regulates Notch receptor trafficking via Rab5-mediated endosomal sorting pathway for Notch activation. These studies support the idea that Notch activation at the plasma membrane is not the only way to transduce Notch signaling, but the Notch receptor must enter through an epsin-mediated endocytic pathway into subcellular compartments to be activated, at least in some contexts.Item Identification and Characterization of Effectors/Binding Molecules for the Small GTPase Rab15(2005-03-18) David Jay Strick; Lisa A. Elferink, Ph.D.; Pomila Singh, Ph.D.; Ping Wu, M.D. Ph.D.; Nancy K. Wills, Ph.D.; Mary L. Thomas, Ph.D.; Gregg T. Nagle, Ph.D.; Brian J. Knoll, Ph.D.Endocytic trafficking is a key mechanism for regulating receptor availability on\r\nthe plasma membrane as well as receptor degradation. Clathrin-dependent endocytosis\r\ninvolves receptor internalization into early endosomes. Here internalized receptors are\r\nsorted for degradation in lysosomes, direct recycling back to the cell surface or indirect\r\nrecycling via a second recycling compartment called the pericentriolar recycling\r\nendosome. Rab GTPases regulate specific membrane trafficking steps including vesicle\r\nbudding, vesicle transport and fusion with downstream target compartments. Rab\r\nfunction is mediated by the cyclical binding and hydrolysis of GTP, which in turn\r\nregulates the recruitment of downstream effector molecules directly involved in\r\nmembrane transport steps. This dissertation focuses on the endocytic GTPase Rab15.\r\nRab15 localizes to early and pericentriolar recycling endosomes, and differentially\r\nregulates receptor transport at these distinct organelles. For example, over expression of\r\nGTP-bound Rab15 inhibits internalization of the Transferrin Receptor and inhibits\r\nhomotypic endosome fusion in vitro. Conversely, over expression of Rab15-GDP\r\ndifferentially stimulates Transferrin receptor recycling from the early endosome and\r\npericentriolar recycling endosome respectively. Rab15 may differentially regulate\r\nreceptor trafficking through these distinct endocytic compartments by binding\r\ncompartment specific effectors. To test this hypothesis, I performed yeast two-hybrid\r\nscreens to identify and characterize Rab15 binding partners. This dissertation is the\r\nfunctional characterization of three Rab15 binding proteins; Mammalian Suppressor of\r\nSec4, Rab15 Effector Protein and Rab15 Binding Protein. Using molecular, biochemical\r\nand imaging approaches, I demonstrated that interactions between Rab15 and Mss4\r\nmodulate the inhibitory effect of Rab15-GTP on receptor entry into early endosomes.\r\nThe second binding partner, Rab15 Effector Protein, localized specifically to the\r\npericentriolar recycling endosome where it regulated Transferrin receptor recycling back\r\nto the cell surface. Finally, Rab15 Binding Protein is a neural specific protein of\r\nunknown function, suggesting an important regulatory function for Rab15 in neural\r\nreceptor trafficking. These results confirm that Rab15 is a bi-functional GTPase, which\r\ndifferentially regulates receptor trafficking through early and pericentriolar recycling\r\nendosomes, by binding specific effector proteins. Moreover, identification of putative\r\nRab15 effector molecules further defines the endocytic pathway, thus providing valuable\r\ninformation for the characterization of trafficking-related diseases and potential drug\r\ntargets in the future.Item Regulation of hepatocyte growth factor receptor endocytic trafficking(2006-03-21) Ning Li; Stan Watowich, Ph.D.; Sarita Sastry, Ph.D.; Lisa A. Elferink, Ph.D.; Cornelis Elferink, Ph.D.; Andrew Bean, Ph.D.The Hepatocyte Growth Factor Receptor (HGFR/cMet) is a receptor tyrosine kinase that is essential for multiple cell responses, including cell proliferation, survival, motility and branching morphogenesis. Normal HGFR signaling regulates embryonic development, organ regeneration and wound healing, whereas deregulated HGFR signaling is linked to tumor progression, metastasis and angiogenesis. Ligand activation of HGFR leads to receptor downregulation via endocytosis and lysosomal degradation, the major mechanism for terminating HGFR signaling. Perturbation of HGFR trafficking, either at the level of internalization or during sorting at the early endosome for degradation, leads to oncogenic activation of HGFR. Impaired HGFR trafficking is emerging as a key mechanism for HGFR-induced cancer progression and metastasis. \r\nA major goal of my dissertation was to lay the foundation for future studies examining different mechanisms leading to altered HGFR trafficking in human cancers, by determining and characterizing the mechanisms which normally function to regulate HGFR internalization and degradation. Two ligands have been identified for HGFR, the physiological ligand HGF and the Listeria surface protein Internalin B (InlB). I characterized the mechanisms for InlB and HGF induced HGFR trafficking using a combination of imaging, molecular biological and biochemical approaches. First, I demonstrated that InlB and HGF were mechanistically equivalent in triggering HGFR internalization primarily through clathrin-coated pits. Then, I determined that the Y1349 and Y1356 docking sites and tyrosine kinase activity of HGFR were required for receptor internalization. Recruitment of the adaptor protein Grb2, but not Gab1, was essential for ligand induced HGFR internalization. I then showed Cbl, an E3 ubiquitin ligase recruited by Grb2 to HGFR, played an essential role in receptor internalization. Furthermore, the E3 ligase activity of Cbl and ubiquitination machinery was involved in HGFR internalization. Finally, I demonstrated that ligand induced HGFR degradation occurred through the lysosomal pathway, involving the function of Hrs and PI3K. My studies represent the first detailed characterization of the trafficking events that normally function to inactivate HGFR signaling. My findings contribute to better understanding of how HGFR-induced tumorigenesis and tumor metastasis may result from impaired HGFR trafficking, and identify novel mechanisms that may function as therapeutic targets for treatment of human cancers due to impaired HGFR trafficking.\r\n