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UNIVERSITY of CALIFORNIA, SAN DIEGO Structural Details And UNIVERSITY OF CALIFORNIA, SAN DIEGO Structural details and mechanism of filamentous actin organization by the isoforms vinculin and metavinculin A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular Pathology by Maria Elisabeth Janssen Committee in charge: Professor Dorit Hanein, Chair Professor Sanford Shattil, Co-Chair Professor Timothy Baker Professor Mark Ginsberg Professor Robert Liddington Professor Robert Ross 2010 Copyright Maria Elisabeth Janssen, 2010 All rights reserved The dissertation of Maria Elisabeth Janssen is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _________________________________________ _________________________________________ _________________________________________ _________________________________________ _________________________________________ Co-Chair _________________________________________ Chair University of California, San Diego 2010 iii Table of Contents SIGNATURE PAGE…………………………………………………………………. iii TABLE OF CONTENTS…………………………………………………………….. iv LIST OF ABBREVIATIONS………………………………………………………… ix LIST OF FIGURES…………………………………………………………………. x LIST OF TABLES………………………………………………….………….……. xiii ACKNOWLEDGEMENTS…………………………………………………………. xiv VITA………………………………………………………………………………….. xv ABSTRACT OF THE DISSERTATION…………………………………………… xvii Chapter 1…………………………………………………………………………….. 1 Background: Vinculin and metavinculin………... …………………………. …. 1 Localization and function in-vivo…………………………………………………... 1 The biological significance of vinculin and metavinculin..................................... 4 Structure of vinculin and metavinculin……………………………………………... 6 Vinculin and metavinculin activation…………………………………………….… 7 Vinculin and metavinculin in dilated- and hypertrophic cardiomyopathy………. 9 Determining how vinculin and metaviculin organize actin filaments…………… 10 References…………………………………………………………………………… 13 Chapter 2…………………………………………………………………………. ….. 18 Background: Actin and actin binding proteins and their analysis using transmission electron microscopy and image reconstruction iv techniques ……………………………………. ...................................................... 18 Structural details on monomeric and filamentous actin……………………….… 18 Regulation of the actin cytoskeletal organization……………………….……….. 23 F-actin severing proteins……………………………………………………….…… 25 Actin crosslinking proteins…………………………………………………….……. 27 Analysis of crosslinked actin arrays…………………………………………….…. 29 Helical reconstruction technique…………………………………………….…….. 31 Electron tomography………………………………………………………………... 33 References……………………………………………………................................. 37 Chapter 3 Janssen et al. (2006) …………………………………………………. 43 Three -dimensional structure of vinculin bound to actin filaments …….... 43 Summary……………………………………………………………………………… 43 Introduction…………………………………………………………………………… 44 Results……………………………………………………………………………….. 45 3D Reconstruction of F-Actin-Vt Complex……………………………………….. 45 The Molecular Details of the Actin-Vinculin Interface…………………………… 45 Comparison with Previous Mutagenesis Experiments……………………………. 47 Binding of F-actin and α-Catenin to Vinculin……………………………………... 47 F-actin Arrays Crosslinked by Vt and Vt-Dimer Model………………………….. 47 Two-Dimensional Image Analysis………………………………………………… 48 Tomographic 3D Reconstructions………………………………………………… 49 v Biochemical Analysis of Vinculin Deletion Mutants……………………………… 49 Discussion…………………………………………………………………………… 50 Mechanism of Actin-Vinculin Binding……………………………………………… 50 Mechanism of Vinculin Dimerization………………………………………………... 51 Activation by Combinatorial Input………………………………………………….. 51 Experimental procedures………………………………………………………….. 52 References………………………………………………………………………….. 53 Supplemental data…………………………………………………………………. 55 Chapter 4 …………………………………………...………………………………… 60 Structural details of vinculin activation ………………………………………… 60 Summary……………………………………………………………………………… 60 Introduction………………………………………………………………………….. 61 Experimental Procedures………………………………………………………….. 65 Results……………………………………………………………………………….. 70 Vinculin activation by F-actin and talin VBSs……………………………………... 70 Vinculin mutant T19 activation by F-actin and talin VBS2 or VBS3…………….. 76 Vinculin activation by α-catenin’s CD3-region……………………………………. 80 Filamentous actin cross-linking by V ∆153………………………………………… 83 Discussion…………………………………………………………………………… 89 Full length vinculin activation………………………………………………………. 89 Cross-linking of actin filaments by V ∆153………………………………………… 91 vi References…………………………………………………………………………… 94 Chapter 5 …………………………………………………………………………….. 96 A 68 amino acid insert changes an actin bundling protein into an actin severing protein: actin organization by vinculin and metavinculin………. 96 Summary……………………………………………………………………………… 96 Introduction…………………………………………………………………………... 97 Experimental Procedures…………………………………………………………… 100 Results…………………………………………………………………………………. 107 Differences in the modes by which Vt and MVt organize filamentous actin…… 107 The F-actin-MVt interaction is weaker than the F-actin-Vt interaction………… 110 Reconstructions of F-actin-Vt and F-actin-MVt assemblies suggest why MVt does not bundle actin filaments……………………………………………………. 111 MVt is an actin severing protein…………………………………………………… 112 MVt binding stabilizes individual actin filaments………………………………….. 116 MV ∆153 bundles actin filaments…………………………………………………… 118 The R975W mutation in MVt does not affect actin organization………………… 119 Actin organization in the presence of both MVt and Vt…………………………… 122 Discussion……………………………………………………………………………. 125 Prevention of actin bundling by MVt………………………………………………... 125 Mechanism of actin severing by MVt………………………………………………. 126 Actin organization by full length metavinculin…………………………………….. 129 vii R975W-MVt affects actin organization in a manner similar to wild-type MVt…. 130 Actin organization in the presence of both Vt and MVt…………………………... 131 References…………………………………………………………………………… 133 Chapter 6 …………………………………………………………………………….. 139 Conclusion and future directions ……………………………………………….. 139 Mechanism of actin bundling by the vinculin tail domain………………………... 139 Evaluating the combinatorial input hypothesis to activate vinculin……………. 140 Filamentous actin organization by metavinculin………………………………… 144 References…………………………………………………………………………… 149 viii List of Abbreviations ABP actin binding protein ADP adenosine diphosphate ATP adenosine triphosphate F-actin filamentous actin G-actin monomeric actin CCD charge coupled device CD α-catenin domain CTF contrast transfer function FRET forster resonance energy transfer IEF isoelectric focussing HSP high speed pellet LSP low speed pellet Sup supernatant Vh vinculin head (M)Vt (meta)vinculin tail domain Vfl full length vinculin VBS vinculin binding site 2D two-dimensional 3D three-dimensional ix List of Figures Fig. 1 -1 Schematic representation of Vinculin and Metavinculin ………………. 1 Fig. 1-2 Vinculin is localized at the ends of actin bundles at focal adhesion sites…………………………………………………………………………………..... 4 Fig. 1 -3 Crystallographic model of vinculin in its auto-inhibited state…………... 6 Fig. 1 -4 Alignment of the Metavinculin insert region among several species…. 7 Fig. 2 -1 Structure of monomeric and filamentous actin………………………….. 20 Fig. 2 -2 Structures of actin and diagrams of fundamental reactions…………… 23 Fig. 2 -3 Example of a two-dimensional F-actin array crosslinked by an actin bundler and its diffraction pattern………………………………………….……….. 30 Fig. 2 -4 The principal of single axis tomography…………………………..……... 35 Fig. 3 -1 3D reconstruction of actin filaments decorated with vinculin tail…..….. 45 Fig. 3 -2 Actin and vinculin-tail docking and interaction analysis……………..…. 46 Fig. 3 -3 Correlation between interaction probabilities, mutations, head-tail contacts, and dimer contacts…………………………………………………….….. 48 Fig. 3 -4 Analysis of cosedimentation experiments…………………………….…. 49 Fig. 3 -5 Analysis of actin arrays crosslinked by the vinculin tail……………….... 50 Fig. 3 -6 Hypothetical model of combinatorial activation and subsequent dimerization of vinculin……………………………………………………………..... 52 Fig. 3 -S1 Cosedimentation assays of full-length his-tagged vinculin, α-catenin vinculin binding domain CD3, and F-actin show that F-actin binding activity of vinculin is enhanced in the presence of CD3……………………………………… 55 Fig. 3 -S2 Light scattering experiments indicate that the interaction of Vt with actin is a complex multistep process……………………………………………….. 56 x Fig. 3 -S3 Differential scanning calorimetry (DSC) probes protein conformations in solution…………………………………………………………..... 57 Fig. 3 -S4 Pyrene fluorescence experiments of labeled G-actin in the presence of Vt…………………………………………………………………………………….. 58 Fig. 4 -1 Schematic representation of talin…………………………………………. 70 Fig. 4 -2 Analysis of vinculin activation in the presence of F-actin and VBS3….. 71 Fig. 4 -3 Analysis of vinculin activation in the presence of F-actin and VBS2….. 72 Fig. 4 -4 Visualization of actin-vinculin-VBS3 assemblies……………………….. 73 Fig. 4 -5 Analysis of vinculin assemblies by size-exclusion chromatography….. 74 Fig. 4 -6 Analysis of vinculin activation in the presence of F-actin and VBS3- peptide…………………………………………………………………………………. 75 Fig. 4 -7 Summary of analysis of several vinculin tail mutants………….……….. 76 Fig. 4 -8 Analysis of T19 vinculin mutant…………………………………………… 77 Fig. 4 -9 Analysis of T19 activation in the presence of F-actin and VBS2 or VBS3………………………………………………………………………………..…. 78 Fig. 4 -10 Analysis of vinculin/T19 activation in the presence of F-actin
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