UT Southwestern Graduate School of Biomedical Sciences
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Print theses and dissertations from 1943 to 2004 are located in the Library's Special Collections and Archives (Room E3.314) and are available by appointment. (Note: Former students may request a digitized copy of their work by email, but other users may submit an Interlibrary Loan request.) For more information, contact archives@utsouthwestern.edu.
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Browsing UT Southwestern Graduate School of Biomedical Sciences by Subject "Actins"
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Item Actin Regulatory Dynamics Required for T Cell Activation: A Quantitative and Systems-Level Perspective(2013-02-04) Roybal, Kole Thomas 1982-; Ward, E. Sally; Wülfing, Christoph; Rosen, Michael K.; Pasare, ChandrashekharT cell activation occurs through interaction with an antigen-presenting cell (APC). Upon activation, signaling ensues with the coordination of dozens of diverse signaling molecules in space and time, a feature of cell signaling we call ‘spatiotemporal patterning’. We performed a systems-scale analysis of the spatiotemporal patterning of T cell signaling and have found that it is highly diverse. Over 50 signaling sensors were imaged in live primary T cells activated with APCs under various physiological stimulation conditions, and no two signaling intermediates showed the same dynamic localization. The activation environment controlled spatiotemporal features of T cell signaling and specific spatiotemporal features correlated with efficient T cell activation. To identify underlying cell biological mechanisms controlling spatiotemporal organization of signaling, we complimented our live cell imaging with microscopy across multiple scales and identified a dense transient F-actin network that extends from a highly interdigitated T cell:APC interface several micrometers deep into the T cell lamellum. Systems-scale imaging revealed a large network of proximal T cell signaling intermediates that localized to the lamellal actin network and shared the spatial, temporal, and mobility features of F-actin. Interference with lamellal actin dynamics modulated the activity of the associated proteins and impaired IL-2 production. These data strongly suggest that the transient deep F-actin network by controlling lamellal localization modulates the activity of a substantial part of the T cell signal transduction system. As a next step in understanding how spatiotemporal dynamics of signaling controls T cell activation, we have developed a quantitative 4D analysis approach for signaling networks and coupled it with traditional cell biological techniques to uncover higher order mechanisms of the control of actin dynamics by CD28 co-stimulation during T cell activation. A group of nine actin regulatory proteins that mediate actin polymerization, capping, and severing were assessed and CD28 co-stimulation was required for their sustained activity at the T cell:APC interface. WAVE2 and Cofilin were especially sensitive to blockade of CD28 signaling. Functional relevance of the loss of WAVE2 and Cofilin enrichment was shown by the treatment of T cells with constitutively active Rac1 and Cofilin, which bypassed the requirement of co-stimulation for normal actin dynamics and AKT activation. This study highlights how a systems analysis of actin regulation could identify mechanisms that are inaccessible to more traditional single protein/gene approaches.Item Biochemical and Cellular Imaging Studies of a Novel CDC42-Dependent Formin Pathway(2006-05-16) Seth, Abhinav; Rosen, Michael K.The Rho GTPases are important regulators of actin cytoskeletal dynamics during processes such as cell migration, cell polarization and cell division. Different Rho family members exert their effects on actin through specific downstream effectors including members of the WASP and Diaphanous-Related Formin (DRF) protein families. It is presently unclear if, and by what mechanisms, the level, timing and localization of Rho GTPase activity control and coordinate effector activity to produce different types of cytoskeletal structures and rearrangements. On a molecular level, autoinhibition is a common regulatory mechanism for many Rho GTPase effectors. Relief of autoinhibition of WASP by the Rho family member Cdc42 involves a significant GTPase-induced conformational change. Based on this conformational change, I have created a series of single-molecule, FRET-based sensors for active Cdc42 that can faithfully report on Cdc42 activity in vitro and in cells. These sensors may be valuable tools for studying the spatio-temporal dynamics of Cdc42 signaling in vivo. The mechanisms of autoinhibition and activation are less well understood for the DRF family of GTPase effectors. DRFs are characterized by a C-terminal Diaphanous Autoregulatory Domain (DAD) that is postulated to regulate the actin assembly activity of the adjacent formin homology 2 (FH2) domain through autoinhibitory interactions with an N-terminal regulatory region, although this has only been shown directly for the DRF mDia1. Here, I show that the actin assembly activity of FRLα, a macrophage-specific DRF, is also autoinhibited by its N-terminal domain. In cells, autoinhibitory interactions also block a novel GTPase-independent membrane localization activity of the N-terminal domain in both FRLα and mDia1. Autoinhibitory control of FRLα activity and localization are specifically relieved by Cdc42. Timelapse microscopy was used to address the potential physiological significance of the Cdc42-FRLα interaction during Fc-γ receptor mediated phagocytosis in macrophages, a Cdc42-dependent process. The data show that FRLα is required for efficient Fc-γ receptor mediated phagocytosis and that it is recruited to the phagocytic cup by Cdc42. These results suggest mutual autoinhibition of biochemical activity and cellular localization may be a general regulatory principle for DRFs and demonstrate an important role for a novel Cdc42-formin pathway in immune function.Item Biochemical Reconstitution and Functional Characterization of the WAVE Regulatory Complex(2009-09-04) Ismail, Ayman Mohamed; Rosen, Michael K.Members of the Wiskott-Aldrich syndrome protein (WASP) family (WASP, N-WASP, WAVE 1-3) have a central role in the transmission of the extracellular signals to the actin cytoskeleton. These proteins use their C-terminal VCA domain to stimulate the actin-nucleating activity of Arp2/3 complex in response to upstream signals from the Rho family GTPases Cdc42 and Rac1. While WASP regulation by GTPases and kinases is well characterized both biochemically and structurally, little is known about WAVE regulation. WAVE exists as part of a five protein complex termed the WAVE Regulatory Complex (WRC). It consists of WAVE, Sra1, Nap1, Abi2 and HSPC300. Biochemical studies of WRC have been hampered by the difficulty of expressing WRC components in bacterial, insect or yeast expression systems. Baculoviruses yielding high expression of each component of WRC were obtained using a modified pFastBac vector, where translation is driven by the lobster tropomyosin promoter. Co-infection into Sf9 cells allowed efficient expression and purification of WRC and two sub-complexes, Sra-Nap and Abi2-WAVE1-HSPC300. We show that WRC is inactive toward Arp2/3 complex in pyrene based actin assembly assays. However, Abi2-WAVE1-HSPC300 heterotrimer is active and Sra1-Nap1 heterodimer inhibits it suggesting that WRC is autoinhibited. A modified WRC complex, where WAVE1 has a PreScission protease site between its VCA domain (the active domain) and its N-terminus and is lacking the proline rich domain, is also inactive toward Arp2/3 complex. However, upon digestion with PreScission protease, this modified complex becomes active. This suggests that the affinity between VCA and the Sra-Nap heterodimer is inherently weak and the heterodimer requires the linkage provided by the Abi2-WAVE1-HSPC300 heterotrimer to VCA to efficiently inhibit it from activating the Arp2/3 complex. The same results are obtained using all Drosophila components. Finally, Rac1-GTP is able to activate WRC towards Arp2/3. However, no dissociation of the complex is detected upon activation by Rac1. In addition to WRC regulation, we have established the mechanism for hyperactivation of VCA through dimerization. We found that a dimeric VCA construct binds Arp2/3 complex with a two VCAs to one Arp2/3 ratio. The affinity of dimeric VCA for Arp2/3 is at least 100 fold higher than monomeric VCA. That explains the potentiation of VCA toward Arp2/3 observed upon VCA dimerization, and provides a mechanistic framework for a new model of WASP regulation superimposed upon allostery. We have also demonstrated that N-WASP and WRC may be able to form a hetero-VCA dimer through the interaction of Abi2 SH3 domain and N-WASP PRD. Such interaction increases the complexity and the signal integration potential of WASp family proteins.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 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 Physical Studies of Actin Nucleation and Conformational Dynamics(2017-09-06) Zahm, Jacob Aaron; Tomchick, Diana R.; Rosen, Michael K.; Rice, Luke M.; Yu, HongtaoActin is a 42 kilodalton ATPase that exists ubiquitously in eukaryotic cells. Unlike other ATPases, however, actin, under suitable conditions, can polymerize, forming helical filaments. Cells, in orchestrating their myriad cellular processes, utilize actin's intrinsic capacity to polymerize, but do so in a tightly controlled fashion, such that new filaments only appear when and where the cell needs them to suit specific purposes. Such control exists at two different levels. Firstly, the stability of actin filaments is subject to "intrinsic" control arising from the state of bound nucleotide. ATP binding favors incorporation of actin monomers into filaments. This incorporation augments actin's ATP hydrolysis activity, and the conversion of ATP to ADP in the nucleotide binding cleft considerably destabilizes filaments, facilitating the return of filament subunits to free monomers. The structural mechanism through which nucleotide conveys information throughout the actin monomer to influence polymerization behavior remains poorly understood and represents a persistent fundamental biological question. In this work I, for the first time, apply modern muti-resonance NMR methods to begin to answer these questions. In addition to the aforementioned intrinsic control, cellular actin is subject to "extrinsic" control via the action of nucleation factors. In order to form a growing filament, actin must proceed through a nucleation step in which monomers must assemble into a thermodynamically and kinetically disfavored nucleus, which ultimately proceeds to a growing filament. Nucleation factors accelerate the rate of filament formation by binding to actin monomers and arranging them into the prerequisite nucleus. In this work, I reveal the crystal structure of actin monomers in complex with the bacterially derived nucleation factor, VopL. The structure represents the first high resolution snapshot of a filament-like nucleation intermediate, and reveals general principles underlying the action of nucleation factors.Item Regulation of Mical Redox Post-Translationally-Driven F-Actin Cytoskeletal Dynamics(2018-04-16) Yesilyurt, Hunkar Gizem; Hibbs, Ryan E.; Terman, Jonathan R.; Rosen, Michael K.; Kavalali, Ege T.The actin cytoskeleton is critical for multiple diverse cellular behaviors, including the ability of an axon to form, extend, navigate, and synapse with its target. Therefore, an important goal is to understand the mechanisms that regulate it. We have been studying one of the largest families of extracellular repulsive guidance cues, the Semaphorins, which were identified in part based on their ability to dramatically dismantle F-actin. More recently, we identified a new actin regulatory protein Mical, which directly associates with both the Semaphorin receptor Plexin and F-actin to post-translationally oxidize actin on its conserved methionine-44 and methionine-47 residues, inducing both F-actin disassembly and altered actin polymerization. Our work has also revealed that this Mical-mediated actin regulatory process is reversible by a specific methionine sulfoxide reductase enzyme called SelR/MsrB. Thus, we have identified an unusual new actin regulatory system - which I sought for my dissertation research to focus on better understanding. I now find that each human MICAL family member, hMICAL-1-3, similar to Drosophila Mical, directly induces F-actin dismantling and controls F-actin-mediated cellular remodeling. Thus, the MICALs are an important phylogenetically-conserved family of catalytically-acting F-actin disassembly factors. I also investigated how this new actin regulatory system fits with classically-studied actin regulatory proteins. Employing a simple biochemical screen, I identified two proteins - cofilin and tropomyosin - that modulate Mical-mediated F-actin disassembly. Further investigation revealed that Mical synergizes with cofilin to rapidly and efficiently dismantle F-actin in a redox regulated manner and that this synergism is also necessary and sufficient for F-actin disassembly in vivo - for remodeling cells, wiring the nervous system, and orchestrating Semaphorin/Plexin repulsion. In contrast, I find that tropomyosin - known to decorate F-actin within specific cellular compartments and at different developmental stages ¬- protects F-actin from Mical-mediated disassembly by stabilizing Mical-oxidized F-actin. Likewise, changing the levels of tropomyosin in vivo results in similar alterations to Mical-mediated F-actin/cellular remodeling suggesting a previously unknown mechanism controlling the plasticity of the actin cytoskeleton with important tissue-specific and developmental/age-related connotations. Thus, my findings provide new insights into the workings of this MICAL-mediated reversible Redox actin regulatory system including its importance to cell, developmental, and neural biology.Item Spatiotemporal Control of Actin Cytoskeletal Machinery in Cells: Multivalency Mediated Protein Clustering at the Plasma Membrane and Optogenetic Tool Development(2015-07-29) Kim, Soyeon; Luby-Phelps, Katherine; Rosen, Michael K.; Yu, Hongtao; Altschuler, Steven J.Precise regulation of actin cytoskeleton dynamics is crucial for numerous cellular processes. My thesis work has demonstrated two different mechanisms that cells use to achieve spatiotemporal control of actin assembly -- multivalency mediated phase separation and subcellular activation of actin polymerization machinery. The plasma membrane of the living cell is believed to function as a dynamic platform for signal transduction, rather than existing simply as a static barrier between intracellular and extracellular environments. Moreover the membrane is organized into distinct lipid and protein microdomains. Recent work has shown the importance of protein assembly at the membrane in the formation of these domains. However, the mechanisms by which proteins organize at the membrane are largely unknown. Here I demonstrate that multivalent interactions among proteins in the Nephrin/Nck/N-WASP actin-regulatory pathway can drive clustering of the components at the plasma membrane in cells. Generation of the micronsized domains is dependent on the phosphorylation-mediated interaction between the transmembrane protein, Nephrin, and its effectors, Nck and N-WASP, in the cytoplasm. These structures are dynamic and associated with increased actin polymerization activity, suggesting the importance of clustering in local rearrangements of the cytoskeleton. My studies suggest that multivalent interactions between the signaling proteins can, in general, contribute to spatiotemporal regulation of cellular processes by clustering of molecules at the plasma membrane. Precise spatiotemporal regulation of protein activity is a key feature of cellular signaling pathways. While contemporary tools in biology have limited control in space and time, recent developments in optogenetics have demonstrated that light can be used as a general tool to investigate protein function in vivo. Many optogenetic tools, however, indirectly regulate protein activity by modulating expression level or cellular localization. Other optogenetic tools with direct control of protein activity have limited applications. Here I demonstrate a method based on fragment complementation that can be generally applicable to a protein of interest based on the light-dependent interaction of Phytochrome B (PhyB) and phytochrome interacting factor 3 (Pif3). My method allows subcellular control of protein function via light-induced reconstitution of a split protein. In particular, I show that light-dependent activation of SopE, a bacterial GEF protein, induces membrane ruffling in a living cell. Furthermore, as an application to inter-molecular regulation between two different molecules, I induced light-mediated interactions between Rho-GTPase Cdc42 and its effector Wiskott-Aldrich Syndrome Protein (WASp) to promote filopodia formation in HeLa cells. Using these optogenetic tools, I demonstrate a general method that can be used to study a broad range of proteins and protein-protein interactions.