Browsing by Subject "endocytosis"
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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 Endocytotic formation of vesicles and other membranous structures induced by Ca2+ and axolemmal injury(Society for Neuroscience, 1998-06-01) Eddleman, Christopher S.; Ballinger, Martis L.; Smyers, Mark E.; Fishman, Harvey M.; Bittner, George D.; Ballinger, Martis L.; Smyers, Mark E.; Bittner, George D.Vesicles and/or other membranous structures that form after axolemmal damage have recently been shown to repair (seal) the axolemma of various nerve axons. To determine the origin of such membranous structures, (1) we internally dialyzed isolated intact squid giant axons (GAs) and showed that elevation of intracellular Ca21 .100 uM produced membranous structures similar to those in axons transected in Ca21-containing physiological saline; (2) we exposed GA axoplasm to Ca21- containing salines and observed that membranous structures did not form after removing the axolemma and glial sheath but did form in severed GAs after .99% of their axoplasm was removed by internal perfusion; (3) we examined transected GAs and crayfish medial giant axons (MGAs) with time-lapse confocal fluorescence microscopy and showed that many injuryinduced vesicles formed by endocytosis of the axolemma; (4) we examined the cut ends of GAs and MGAs with electron microscopy and showed that most membranous structures were single-walled at short (5–15 min) post-transection times, whereas more were double- and multi-walled and of probable glial origin after longer (30–150 min) post-transection times; and (5) we examined differential interference contrast and confocal images and showed that large and small lesions evoked similar injury responses in which barriers to dye diffusion formed amid an accumulation of vesicles and other membranous structures. These and other data suggest that Ca21 inflow at large or small axolemmal lesions induces various membranous structures (including endocytotic vesicles) of glial or axonal origin to form, accumulate, and interact with each other, preformed vesicles, and/or the axolemma to repair the axolemmal damage.Item Regulation of the contractile vacuole in Dictyostelium amoebae(2012) Lu, Steve; De Lozanne, ArturoThe contractile vacuole (CV) is a protozoan organelle network that is responsible for osmoregulation by actively expelling water from the cell in hypotonic environments. However the mechanism by which the CV interacts with the plasma membrane to discharge its contents is poorly understood. While little is known about the biogenesis of this organelle, knockout studies in Dictyostelium discoideum have demonstrated the requirement of several proteins such as clathrin for normal CV formation. However, clathrin-mediated endocytosis may not be the directly responsible for the organization and recycling of CV components. With fluorescence microscopy and fluorescently-labeled proteins, 1-butanol, a reversible inhibitor of clathrin function, was applied to observe the direct role of clathrin in CV function. The involvement of actin was also examined by blocking polymerization with latrunculin. While actin inhibition does not immediately perturb CV function, 1-butanol treatment results in a rapid widespread disruption of the CV network. Because 1-butanol is a known PLD inhibitor, phosphatidic acid, the immediate product of PLD, was applied in the presence of 1-butanol. Phosphatidic acid resulted in only modest recovery, suggesting the fine balance in signaling required for appropriate CV function. These results implicate the role of PLD activity in CV assembly and maintenance. Understanding the pathway by which CV function is regulated will yield insights into the function of proteins in the endocytic pathway as well as provide a framework for similar studies in human macrophages.