Browsing by Subject "Actin Cytoskeleton"
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Item Functions of Phosphatidylinositol 4-Phosphate 5 Kinases in Actin Cytoskeletal Regulation during Phagocytosis(2009-06-18) Mao, Yuntao; Yin, Helen L.Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a crucial signaling phosphoinositide at the plasma membrane (PM) which mediates a variety of biochemical activities and cellular functions. It is primarily synthesized by type I phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) through the phosphorylation on the D-5 position of the inositol ring of phatidylinositol 4-phosphate [PI(4)P]. Mammals have three PIP5K isoforms named a, b, and g (human isoform designation) which have a highly conserved central kinase homology domain and divergent amino and carboxyl terminal extensions. There is now extensive evidence suggesting that PIP5Ks have unique functions and regulations in many cellular processes which provide the key to understand how functionally, and possibly physically, segregated PIP2 pools are generated. The actin cytoskeleton is dynamically remodeled during Fcg receptor (FcgR)-mediated phagocytosis in a PIP2-dependent manner. I investigated the role of PIP5Kg and a isoforms, which synthesize PIP2, during phagocytosis. PIP5Kg-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgR clustering. Delivery of exogenous PIP2 rescued these defects. PIP5Kg knockout BMM also have more RhoA and less Rac1 activation and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5Kg by RNA interference (RNAi) inhibits particle attachment. In contrast, PIP5Ka knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein (WASP) activation and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5Kg but not a is transiently activated by spleen tyrosine kinase (Syk)-mediated phosphorylation. I propose that PIP5Kg acts upstream of Rac/Rho and that the differential regulation of PIP5Kg and a allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.Item The Role of the PIP5 Kinase Gamma 87 Isoform in the Regulation of the Actin Cytoskeleton(2010-01-12T18:49:50Z) 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.