Browsing by Subject "Actin Cytoskeleton"
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Item Direct Redox Regulation of F-Actin Assembly and Disassembly by MICAL(2013-04-08) Hung, Ruei-Jiun 1982-; Hiesinger, Peter Robin; Terman, Jonathan R.; Rosen, Michael K.; Yin, Helen L.How guidance cues present outside of cells exert their precise effects on the internal actin cytoskeleton is poorly understood. Such effects are critical for diverse cellular behaviors including polarity, morphology, adhesion, motility, process elongation, navigation, and connectivity. Semaphorins, for example, are one of the largest families of these guidance signals and play critical roles in neurobiology, angiogenesis, immunology, and cancer. One interesting characteristic of the Semaphorins is that they inhibit the movement of cells (and their membranous processes) through their ability to disrupt actin cytoskeletal organization. However, despite considerable progress in the identification of Semaphorin receptors and their signal transduction pathways, the molecules linking them to the precise control of the actin cytoskeleton have remained mysterious. During my graduate studies, I sought to better understand a family of unusual proteins called the MICALs (which includes one Drosophila Mical and three vertebrate MICALs), which associate with the Semaphorin cell-surface receptor Plexin and are important for Semaphorins to exert their effects. Nothing was known, however, regarding the specific role of the MICALs in these Semaphorin-dependent events. Not long after I began my graduate work, my colleagues and I noticed that Mical was necessary for proper actin cytoskeletal organization and sufficient to reorganize the actin cytoskeleton in vivo. Therefore, to better understand the role that Mical plays in actin cytoskeletal rearrangements, I took a biochemical approach, and purified the Mical protein. Utilizing biochemical and imaging approaches with purified proteins, I found that Mical directly binds to actin filaments (F-actin) and is able to induce the rapid disassembly of F-actin. Thus, my results revealed that Mical is a novel F-actin disassembly factor that provides a molecular conduit through which F-actin disassembly can be precisely achieved in response to Semaphorins. So I next wondered how Mical induces F-actin disassembly. Interestingly, the MICALs belong to a class of flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in oxidationreduction (Redox) reactions. Although MICALs have no known substrate/s, my in vivo and in vitro results revealed that Mical employs its Redox region to bind F-actin and disassembles filaments in an NADPH-dependent manner. Moreover, this Mical-treated actin failed to repolymerize even after removal of Mical, indicating that Mical stably modifies actin to alter polymerization. Mass spectrometric analyses revealed that F-actin subunits were directly modified by Mical on their conserved pointed-end that is critical for filament assembly. Specifically, Mical post-translationally oxidized a conserved amino acid (Methionine 44) within a region of actin that is critical for actin-actin contacts, simultaneously severing filaments and decreasing polymerization. Thus, my thesis observations reveal a novel and specific oxidation dependent signaling mechanism that selectively regulates actin dynamics and cellular behaviors.Item Functions of Phosphatidylinositol 4-Phosphate 5 Kinases in Actin Cytoskeletal Regulation During Phagocytosis(2009-06-18) Mao, Yuntao Steve; 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 Molecular Basis of Tissue Elasticity and Force Balance During Drosophila Gastrulation(December 2021) Goldner, Amanda Nicole; Collins, James J.; Doubrovinski, Konstantin; Chen, Elizabeth; Douglas, PeterThe mechanics of folding any material rely on two things: the physical forces forming the fold and the material properties of the substance being folded. When working in biological tissue such as an early Drosophila embryo, there is no existing way to directly measure morphogenetic forces, and the relative contributions of forces in various cellular domains remain unknown. To begin, I studied gastrulation in a genetic background where basal membranes never form and cells remain open to the yolk sack throughout the course of VF formation. Strikingly, the VF is still capable of forming in this background. I extensively characterize this phenotype by a combination of electron microscopy and immunofluorescence. My observations rule out a class of popular models of VF formation that would generically predict no folding in the absence of basal membranes. To address this discrepancy, we propose that viscous shear forces play a major role in allowing the furrow to form. We have developed a new computational model that takes cytoplasmic viscous shear into account. In accordance with our observations, our model predicts that basal membranes are dispensable for VF formation. Tissue material properties such as elasticity are also key to fold shape. In vivo tissue deformation experiments show that embryonic tissue is elastic in the stages leading up to gastrulation. Inhibiting F-actin polymerization severely decreases elasticity. I propose that different characteristics of F-actin networks - e.g. branching, remodeling, and crosslinking - are variably responsible for conferring elasticity. It is unclear whether the presence of active forces along actin filaments contributes to tissue elasticity. To this end, I engineered the auxin-inducible degron system to degrade the foremost source of active forces in F-actin networks: myosin II. My design allows us to specifically degrade Drosophila myosin II in under 1hr in vivo. This will allow us to precisely quantify the contribution of myosin II to not only tissue elasticity, but any other feature or developmental process of interest.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.