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Structural Characterization of Two Different F- Actin Cross-Linking Proteins by Electron Microscopy Cheri M

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Structural Characterization of Two Different F- Actin Cross-Linking Proteins by Electron Microscopy Cheri M Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2006 Reading Between the Filaments: Structural Characterization of Two Different F- Actin Cross-Linking Proteins by Electron Microscopy Cheri M. Hampton Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES READING BETWEEN THE FILAMENTS: STRUCTURAL CHARACTERIZATION OF TWO DIFFERENT F-ACTIN CROSS-LINKING PROTEINS BY ELECTRON MICROSCOPY By CHERI M. HAMPTON A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Fall Semester, 2006 The members of the Committee approve the Dissertation of Cheri M. Hampton defended on August 11th, 2006. _________________________ Kenneth A. Taylor Professor Directing Dissertation _________________________ Charles Ouimet Outside Committee Member _________________________ P. Bryant Chase Committee Member _________________________ Piotr Fajer Committee Member _________________________ Hong Li Committee Member Approved: _________________________ Timothy M Logan, Director, Institute of Molecular Biophysics The Office of Graduate Studies has verified and approved the above named committee members. ii TABLE OF CONTENTS List of Tables .................................................................................... vi List of Figures .................................................................................... vii Abstract .......................................................................................... x 1. Introduction to Actin Cross-linking Proteins and EM.......................... 1 1.1 The Cytoskeleton is a Cellular Scaffold................................... 1 1.1.1 Actin Filaments in the Cytoskeleton ................................. 1 1.1.2 Cell Structure and Tension .............................................. 2 1.1.3 Cell Structure and Signaling............................................. 3 1.2 Actin, Function and Structure .................................................. 6 1.2.1 Actin is the Most Highly Conserved Protein ..................... 6 1.2.2 Actin Function .................................................................. 6 1.2.3 Actin Structure ................................................................. 7 1.3 The Spectrin Repeat Family, Function and Structure.............. 10 1.3.1 Spectrin Repeat Family.................................................... 10 1.3.2 α-Actinin Function ............................................................ 14 1.3.3 α-Actinin Structure ........................................................... 17 1.4 The Gelsolin Family, Function and Structure .......................... 19 1.4.1 Actin Filament Severing, Capping, Nucleating,................ and Bundling Proteins....................................................... 19 1.4.2 Villin Found Primarily in Microvilli..................................... 19 1.4.3 Villin is Structurally Similar to Gelsolin ............................. 20 1.4.4 Gelsolin is Regulated by Calcium .................................... 26 1.5 Electron Microscopy, Electron Tomography,........................... And Image Analysis ................................................................. 28 1.5.1 Electron Microscopy and Image Formation...................... 28 1.5.2 The Fourier Transform ..................................................... 29 1.5.3 The Contrast Transfer Function ....................................... 30 1.5.4 Tomography and the Projection Theorem........................ 31 1.5.5 Averaging and Classification............................................ 32 2. α-Actinin is a Variable-Length Actin Cross-linker .............................. 34 2.1 Background ............................................................................. 34 iii 2.2 Results .................................................................................... 37 2.2.1 Alignment and Correspondence Analysis ........................ 37 2.2.2 Distribution of Cross-Link Angles and Lengths ................ 44 2.2.3 α-Actinin Binds to a Single Actin Filament ....................... 49 2.3 Discussion............................................................................... 51 2.3.1 α-Actinin-Membrane Interactions ..................................... 52 2.3.2 Variability of Bipolar Cross-Links ..................................... 53 2.3.3 Significance of “Monofilament Binding”............................ 54 2.3.4 Significance of Variable Length of Cross-Link.................. 56 2.4 Materials and Methods............................................................ 57 2.4.1 Protein Purification........................................................... 57 2.4.2 EM Data Collection .......................................................... 58 2.4.3 Image Processing ............................................................ 58 2.4.4 Classification.................................................................... 58 2.2.5 Modeling .......................................................................... 59 3. α-Actinin Interacting Proteins ............................................................ 61 3.1 The Alliance for Cellular Signaling........................................... 61 3.2 Molecule Page Navigation....................................................... 62 3.3 α-Actinin-1 Molecule Pages .................................................... 68 3.3.1 α-Actinin-1 Overview........................................................ 68 3.3.1 α-Actinin-1 Mini Molecule Page ....................................... 73 3.4 α-Actinin-2 Molecule Pages .................................................... 77 3.4.1 α-Actinin-2 Overview........................................................ 77 3.4.2 α-Actinin-2 Summary ....................................................... 79 3.4.3 α-Actinin-2 Network Map ................................................. 93 3.4.4 α-Actinin-2 States ............................................................ 94 3.4.5 α-Actinin-2 Transitions ..................................................... 96 3.5 α-Actinin-3 Molecule Pages .................................................... 98 3.5.1 α-Actinin-3 Overview........................................................ 98 3.5.2 α-Actinin-3 Summary ....................................................... 100 3.5.3 α-Actinin-3 Network Map ................................................. 104 3.5.4 α-Actinin-3 States ............................................................ 105 iv 3.5.5 α-Actinin-3 Transitions ..................................................... 106 3.6 α-Actinin-4 Molecule Pages .................................................... 107 3.6.1 α-Actinin-4 Overview........................................................ 107 3.6.2 α-Actinin-4 Summary ....................................................... 109 3.6.3 α-Actinin-4 Network Map ................................................. 118 3.6.4 α-Actinin-4 States ............................................................ 119 3.6.5 α-Actinin-4 Transitions ..................................................... 121 3.7 α-Actinin Interacting Proteins .................................................. 123 4. Villin in an Unusual Suspect in F-actin Cross-Linking ....................... 135 4.1 Background ............................................................................. 135 4.2 Results .................................................................................... 138 4.2.1 Image Alignment and Volume Classification.................... 138 4.2.2 Models ............................................................................. 143 4.3 Discussion............................................................................... 146 4.3.1 A Tool for Studying Actin: Actin-binding Protein Interactions 147 4.3.2 Significance of Villin-Actin Interaction Sites ..................... 147 4.3.3 Indication for Multiple Modes of F-actin Binding............... 148 4.4 Materials and Methods............................................................ 149 4.4.1 Protein Purification and Array Formation ......................... 149 4.4.2 EM Data Collection .......................................................... 149 4.4.3 Image Processing ............................................................ 150 4.4.4 Classification.................................................................... 150 4.4.5 Modeling .......................................................................... 150 5. Summary and Future Directions........................................................ 152 REFERENCES .................................................................................... 154 BIOGRAPHICAL SKETCH .................................................................... 171 v LIST OF TABLES Table 2.2.1.1: Assessment of Classification Accuracy ........................... 43 Table 3.7.1: α-Actinin Interacting Proteins ............................................. 123 vi LIST OF FIGURES Figure 1.1.1.1: Z Disc Architecture ........................................................ 2 Figure 1.1.2:1: The Cytoskeletal Network ............................................. 3 Figure 1.1.3:1: The Focal Adhesion is a Site for Force-Regulated Cell Signaling .................................. 5 Figure 1.1.3.2: Schematic of Protein Interactions Emanating from the Z Disc
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