Browsing by Subject "Endocytosis"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Cellular determinants of reovirus entry and infection
    (2012-10) Schulz, Wade Loren
    Efficient entry is a critical determinant of reovirus oncolysis. While a model for the entry of reovirus virions has been described, the method by which partially uncoated intermediate subviral particles (ISVPs) enter host cells remains unknown. Biochemical studies have suggested that ISVPs can directly penetrate the plasma membrane since these particles can cause membrane damage and hemolysis at high multiplicities of infection. We used a combination of techniques to show that these particles can use endocytosis to enter and productively infect host cells. We found that uptake of reovirus virions and ISVPs was inhibited in cells treated with a small molecule inhibitor of dynasore. Analysis of reovirus replication in cells treated with genistein and in cells expressing dominantnegative caveolin-1 revealed that ISVPs use dynamin-dependent caveolar endocytosis to enter host cells. These studies also showed that reovirus virions were able to take advantage of caveolar endocytosis to infect cells. Because many viruses have recently been found to enter cells through dynamindependent and –independent endocytic pathways, we also assessed the role of cholesterol in reovirus infection. The growth of reovirus virions, but not ISVPs, was inhibited in cells treated with methyl-β-cyclodextrin, which extracts membrane cholesterol. We also wanted to learn whether reovirus entry had cell type-specific differences. Analysis of reovirus infection in a rat embryo fibroblast (CREF) and Ras-transformed CREF cell line suggested that reovirus particles lose the ability to use clathrin-mediated endocytosis in CREF cells following Ras transformation. Reovirus can also infect cells that lack caveolae. To assess whether reovirus used different endocytic pathways to infect cells devoid of caveolae, we analyzed reovirus entry and infection in polarized Caco-2 cells. While reovirus infection of these cells was dynamin-independent, particle internalization occurred through both dynamin-dependent and –independent pathways. Visualization of particle uptake showed that particles internalized through dynamin-dependent endocytosis were transcytosed to the basolateral surface. These findings have demonstrated that reovirus can use a diverse set of endocytic pathways to enter and infect cells. The data presented in this thesis show that reovirus entry is a complex process that can vary in different cell types. In addition, it has revealed that particles internalized into various endocytic pathways are trafficked to different destinations in polarized epithelial cells.
  • Thumbnail Image
    Item
    Identifying the mechanism of action of the antiepileptic drug levetiracetam in synaptic vesicle release and its implications for epilepsy.
    (2011-07) Meehan, Anna L.
    In the United States alone, over 3 million people have been diagnosed with epilepsy, a dynamic disease characterized by recurring and unpredictable seizures. Antiepileptic drugs (AEDs) and other therapies have helped to combat this widespread phenomenon, yet for one-third of epilepsy patients, there is still no effective treatment. A better understanding of the mechanisms of AEDs has been called for. Levetiracetam (LEV) is one AED on the market that does not work by the typical mechanisms of action of AEDs. LEV binds to the vesicular protein SV2A in neurons in the brain, which is thought to mediate some step in neurotransmitter release. However, the exact mechanism of action of LEV is unknown. Deducing this mechanism would be of substantial benefit for the development of new, similar drugs that also work by such a non-conventional mechanism. The experiments and results I present in this dissertation detail my investigation of the effect of LEV on neurotransmission of rat hippocampal neurons. My methods include fluorescence-staining with FM dyes to label synaptic vesicles and monitor vesicle release as well as electrophysiological techniques to observe synaptic currents. My experimental protocols have allowed me to detect differences in neurotransmitter release in LEV-treated neurons. Furthermore, manipulating exposure conditions required before LEV action have allowed me to deduce that LEV must enter active neurons to reach SV2A. I believe that LEV is dependent on endocytosis for access to vesicles, a completely unique mechanism of action for a small molecule drug.
  • Thumbnail Image
    Item
    The role of receptor transport in notch signaling.
    (2010-12) Sorensen-Kamakian, Erika Beth
    The Notch signaling pathway is important for cell differentiation and proliferation. The tight regulation of Notch signaling is critical, as aberrant signaling is associated with human cancers. Recent observations suggest that endocytosis is critical in modulating both the activation and down-regulation of Notch signaling events. Although endocytosis is clearly important, it is unclear how endocytosis regulates the Notch signaling pathway. To directly investigate the relationship between Notch internalization and signaling, we developed a quantitative Notch uptake assay using mammalian tissue culture cells. siRNA-mediated depletion studies reveal that Notch endocytosis is clathrin-dependent and requires epsin1, AP2, and Nedd4. Moreover, we show by coimmunopreipitation analysis that epsin1 interaction with Notch is ubiquitin-dependent. Contrary to current models, we demonstrate, by reporter assay, that internalization defects lead to elevated Notch signaling. These results indicate that signal activation occurs independently of Notch endocytosis. In addition to identifying endocytic components critical to regulating Notch signaling, we evaluated the relationship between AAK1 and Numb, two conserved factors implicated in Notch signaling. We find that AAK1, a key endocytic kinase, interacts with and phosphorylates Numb. Mutation of the AAK1 phosphorylation site restricts Numb localization and changes Numb migration when resolved by SDS-PAGE. To test if Numb phosphorylation plays a role in receptor internalization, we employ uptake assays using both immunofluoroscence and a quantitative Elisa-based approach. We find that overexpression of mutant Numb potently disrupts transferrin receptor internalization. Collectively, these observations indicate that phosphorylation is a general mechanism that regulates Numb activity by modulating its distribution within the cell.