Browsing by Subject "Phosphatidylinositols"
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Item Illuminating Organellar and Molecular Organization of the Endomembrane System(2018-07-12) Hsieh, Ting-Sung; Schmid, Sandra; Goodman, Joel M.; Jaqaman, Khuloud; Liou, JenThe endomembrane system consists of virtually all of the structurally and functionally distinct membrane compartments in a eukaryotic cell, except mitochondria and plastids. Different compartments in the endomembrane system communicate with each other through vesicular transport as well as direct membrane-membrane contact. In this work, I first probe spatial organization of membrane contact sites between the endoplasmic reticulum and the plasma membrane in mammalian cells. These membrane contact sites, termed endoplasmic reticulum-plasma membrane junctions, mediate cellular activities ranging from Ca2+ signaling to lipid metabolism. I provide quantitative information on spatial organization of endoplasmic reticulum-plasma membrane junctions and show that it is in part regulated by F-actin. This gives clues about how cellular activities of endoplasmic reticulum-plasma membrane junctions may be regulated because their spatial organization dictates the extent and location of these cellular activities. Then, I examine the phosphoinositide identities of mammalian late endocytic compartments. Phosphoinositides perform the pivotal role as identifiers for different membrane compartments. I show that phosphatidylinositol 3-phosphate is presnt on late endosomes while phosphatidylinositol 4-phosphate is presnt on some endolysosomes and lysosomes. This yields clues about how phosphoinositides regulate membrane sorting and biogenesis of different late endocytic compartments. Last, I show the alteration of phosphoinositide identity of the endoplasmic reticulum by the phosphatidylinositol 3-kinase MavQ secreted by Legionella pneumophila. L. pneumophila exploits the host endoplasmic reticulum membrane to form a Legionella-containing vacuole for intracellular replication. I reveal that MavQ generates phosphatidylinositol 3-phosphate on the endoplasmic reticulum membrane and that MavQ, together with the phosphatidylinositol 3-phosphatase SidP, self-organizes and propagates on the endoplasmic reticulum membrane in a wave-like manner and drives vesicle/tubule generation along the way. This not only provides insight into how L. pneumophila subverts the host cell to establish their own niche but also highlights the importance of concerted kinase/phosphatase integration in generating complex cellular behaviors.Item Molecular and Functional Analysis of Phosphatidylinositol 4 Kinase Type II Beta(2008-09-12) Jung, Gwanghyun; Albanesi, Joseph P.Phosphoinositides play fundamental roles in controlling membrane-based signaling events. Phosphatidylinositol 4-kinases (PI4Ks) catalyze the production of PI4P, a major precursor in phosphoinositide biosynthesis, and consist of two classes (type II and type III), each divided into two isoforms (alpha and beta ). PI4KIIalpha and beta differ primarily in their distributions between cytosol and membranes: PI4KIIalpha is almost exclusively membrane-bound, by virtue of its palmitoylation in a cysteine-rich motif; although PI4KIIbeta also contains a palmitoylatable cysteine-rich motif, this isoform is almost evenly distributed between membranes and cytosol, and only about half of the membrane-associated pool is palmitoylated. My study focused on determining the functions of post-translational modifications and domains of PI4KIIbeta and on identifying its binding partners, with the long-term goal of understanding the roles and mechanisms of regulation of this kinase. Domain analysis shows that the C-terminal 160 amino acids of PI4KIIs determine the distinct membrane binding properties and activities of PI4KIIalpha and beta . As expected, based on our previous work with PI4KIIalpha , palmitoylation of PI4KIIbeta is important for its membrane binding and activity. Although PI4KIIbeta is also phosphorylated in cells, this modification has no detectable effect on any examined property of the kinase. Immunoprecipitation and mass spectrometry revealed a genuine binding partner for PI4KIIbeta , Hsp90. The functional significance of the Hsp90-PI4KIIbeta interaction was defined using geldanamycin, a specific Hsp90 inhibitor. Geldanamycin treatment disrupts the interaction and destabilizes PI4KIIbeta , reducing its half-life by 40% and increasing its susceptibility to proteasomal degradation. Although full-length PI4KIIalpha does not bind Hsp90, and is not destabilized by geldanamycin treatment, a cytosolic PI4KIIalpha truncation mutant becomes sensitive to geldanamycin and binds to Hsp90. Thus, both PI4KII isoforms contain Hsp90 binding sites but only PI4KIIbeta requires Hsp90 for stabilization, presumably because there is a substantial cytosolic pool of this isoform. Interestingly, brief exposure to geldanamycin causes a partial redistribution of PI4KIIbeta from the cytosol to membranes, which results in increased PI4P synthesis in cells. Moreover, the growth factors EGF and PDGF also disrupt the interaction between Hsp90 and PI4KIIbeta , suggesting that Hsp90 not only protects PI4KIIbeta from degradation, but may also prolong its residency in the cytoplasm until extracellular signals release Hsp90 from the kinase. Currently the precise roles of PI4KIIbeta are unknown. Based on its partial redistribution to the plasma membrane upon cell treatment with growth factors, I speculated that PI4KIIbeta may somehow be involved in receptor-mediated endocytic trafficking. My results, employing siRNA-based knockdown strategies, indicate that depletion of PI4KIIbeta enhances early steps of EGFR internalization and subsequent initiation of ERK activation in response to EGF treatment. The facilitated endocytosis that results from this depletion is likely due to an increase in endosomal fusion. Indeed, activities of the endosomal fusion facilitators EEA1 and Rab5 increase in PI4KIIbeta depleted cells. These results suggest for the first time a role of PI4KIIbeta in endocytic trafficking and signaling of the EGFR.Item The Role of the PIP5 Kinase Gamma 87 Isoform in the Regulation of the Actin Cytoskeleton(2010-01-12) Corgan, Anne Marie; Yin, Helen L.Phosphatidylinositol-4,5-bisphosphate (PIP2) is an important regulator of the actin cytoskeleton and plasma membrane functions. It is primarily synthesized by the type 1 phosphatidylinositol 4 phosphate 5 kinases (PIP5Ks). Mammals have three PIP5K genes (PIP5K alpha, PIP5K beta, and PIP5K gamma), and the gamma isoform has two ubiquitous 90 kDa and an 87 kDa splice variants. We found that the depletion of each PIP5K isoform individually by RNA interference (RNAi) or gene knockout by homologous recombination generated distinct changes in the actin cytoskeleton and signaling responses. The actin phenotype of the PIP5K gamma depletion (using pan siRNA, which is directed against a common sequence shared by the 90 and 87kDa isoforms) in HeLa cells is particularly striking: it results in increased actin stress fibers, decreased chemotaxis, and increased adhesion to fibronectin-coated substrates. There is also a striking increase in prominent focal adhesions (FA). Using real-time IRM, we found that the turnover of FA is 48% slower in the PIP5K gamma depleted cells. Likewise, there is a large decrease in the dynamic turnover of green fluorescent protein (GFP)-labeled vinculin and paxillin in FA, as monitored by fluorescence recovery after photobleaching. Since PIP5K gamma 90 has already been implicated in FA assembly, we depleted it specifically without depletion of the much more abundant PIP5K gamma 87 by using a PIP5K gamma 90 specific targeting sequence not found in PIP5K gamma 87. This fails to produce robust stress fibers. Overexpression of PIP5K gamma 87, but not the kinase dead enzyme, is able to rescue the pan PIP5K gamma knockdown actin phenotype in HeLa cells. Thus, PIP5K gamma 87 is the major contributor to the pan PIP5K gamma depletion/knockout robust actin and FA phenotype. Similar results were obtained in mouse embryonic fibroblasts (MEFs) from PIP5K gamma -/- mice. We sought to identify the molecular mechanisms of the PIP5K gamma depleted actin phenotype. Inhibitors of myosin, Rho-associated coiled-coil-containing protein kinase (ROCK), and RhoA GTPase all decreased the amount of thick actin stress fibers in PIP5K gamma RNAi cells, suggesting that the phenotype is due to abnormal RhoA activation. This is confirmed by the finding that RhoA activity is elevated in PIP5K gamma depleted/knock out cells. We hypothesize that PIP5K gamma regulates the actin cytoskeleton by inhibiting Rho, and thus its downstream effectors ROCK and myosin.