Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1976 Localization of [alpha]-actinin and tropomyosin in muscle and nonmuscle systems Judith Vance Schollmeyer Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biology Commons Recommended Citation Schollmeyer, Judith Vance, "Localization of [alpha]-actinin and tropomyosin in muscle and nonmuscle systems " (1976). Retrospective Theses and Dissertations. 5703. https://lib.dr.iastate.edu/rtd/5703 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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Xerox University iViicrofiims 300 North Zeeb Road Ann Arbor, Michigan 48106 77-1478 SCHOLLMEYER, Judith Vance, 1945- LOCALIZATION OF d-ACTININ AND TROPOMYOSIN IN MUSCLE AiND NONMUSCLE SYSTEMS. lowa State University, Ph.D., 1976 Biology Xerox University iviicrofilms, Ann Arbor, Michigan 48106 @Copyright by JUDITH VANCE SCHOLLMEYER Localization of a-actinin and tropomyosin in muscle and nonmuscle systems by Judith Vance Schollmeyer A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Major; Molecular, Cellular, and Developmental Biology (Animal Science) Approved : Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1976 Copyright©Judith Vance Schollmeyer, 1976. All rights reserved. ii TABLE OF CONTENTS Page LIST OF ABBREVIATIONS xii INTRODUCTION 1 REVIEW OF LITERATURE 4 General Review 4 Z-Disk Ultrastructure 13 Z-Disk Composition 36 Cardiac Muscle 40 Nemaline Myopathy 48 Smooth Muscle 50 The Fibroblast 54 Other Nonmuscle Cells 56 MATERIALS AND METHODS 63 Biochemical Procedures 63 Immunological Procedures 79 Morphological Procedures 116 RESULTS 131 Localization of a-Actinin and Tropomyosin in Three Muscle Types 132 Nemaline Myopathy Muscle 162 Smooth Muscle 173 Fibroblasts 192 Brush Borders of Intestinal Epithelial Cells 209 Acrosomal Process of Limulus Sperm 223 Attempts to Determine the Molecular Architecture of the Z-Disk 237 lii Page Studies on Nemaline Myopathy Muscle 266 Studies on IZI Brushes 274 DISCUSSION 293 Distribution and Location of a^Actinin and Tropomyosin in Biological Movement Systems 293 Molecular Anatomy of the a-Actinin/Actin Interaction and its Implications for Z-Disk and Dense Body Structure 304 What Implications Do the Distribution ai>^ .ocation of a-Actinln and Tropomyosin Have for tb 7siological Functions of these Proteins? 315 SUMMARY AND CONCLUSIONS 327 LITERATURE CITED 335 ACKNOWLEDGEMENTS 354 iv LIST OF FIGURES Figure Page 1. Diagrammatic representation of the Z-disk model proposed by Knappeis and Carlsen (1962) 16 2. Two identical wire models showing structures of Z-disk model proposed by Kelly (1967) 20 3. Diagrammatic representation of Rowe's (Rowe, 1973) looping filament model of Z-disk structure 23 4. Schematic diagrams of cross sections of three different Z-disk models as summarized by Franzini-Armstrong (1973) 32 5. Flow sheet showing preparation of purified myofibrils by using differential centrifugation 65 6. Flow sheet showing preparation of IZI brushes from purified myofibrils and extraction of IZI brushes with 1 M KCl and «2° 69 7. Flow sheet showing preparation from chicken gizzard smooth muscle of contractile filaments swollen at low ionic strength and extraction of a-actinin from these swollen filaments 82 8. Flow sheet showing preparation of a-actinin and tropomyo­ sin from low ionic strength extracts of swollen contractile filaments from chicken gizzard smooth muscle 85 9. SDS-polyacrylamide gel electrophoresis of porcine cardiac, chicken gizzard, and porcine skeletal a-actinins, and of contaminating proteins removed from porcine skeletal a-actinin by preparative electrophoresis 90 10. SDS-polyacrylamide gel electrophoresis of electrophoreti- cally purified porcine cardiac, chicken gizzard, and porcine skeletal a-actinins 92 11. SDS-polyacrylamide gel electrophoresis of electrophoreti- cally purified porcine cardiac, chicken gizzard, and porcine skeletal tropomyosin 94 12. SDS-polyacrylamide gel electrophoresis of contaminating proteins removed from porcine cardiac, chicken gizzard, and porcine skeletal tropomyosins by preparative poly- acrylamide electrophoresis 96 V Figure Page 13. Elution profile of rabbit IgG from a 2.5 x 25 cm DEAE- cellulose column 102 14. Elution profile of papain-treated sheep anti-rabbit IgG from a 2.5 X 30 cm CM-cellulose column 104 15. SDS-polyacrylamide geis of Fab fragments before and after conjugation with peroxidase and microperoxidase 107 16. Elution profile of sheep IgG anti-rabbit IgG conjugated with peroxidase from a 1.6 x 90 cm Sephadex G-200 column 111 17. Elution profile of sheep Fab anti-rabbit IgG conjugated with peroxidase from a 1.6 x 90 cm Sephadex G-200 column 113 18. Elution profile of sheep Fab anti-rabbit IgG conjugated with microperoxidase from a 1.6 x 90 cm Sephadex G-lOO column 115 19. Iinmunoelectrophoretic patterns of different antibody fractions 118 20. Electron micrograph of glycerinated porcine psoas muscle 135 21. Electron micrograph of glycerinated porcine papillary muscle 135 22. Fluorescence and phase micrographs of porcine psoas myofibril after incubation with anti-a-actinin and FITC- labaled sheep anti-rabbit IgG 138 23. Fluorescence and phase micrographs of porcine psoas myofibril after incubation with anti-tropomyosin and FITC-labeled sheep anti-rabbit IgG 138 24. Fluorescence and phase micrographs of porcine papillary myofibril after incubation with anti-a-actinin and FITC- labeled sheep anti-rabbit IgG 141 25. Fluorescence and phase micrographs of porcine papillary myofibril after incubation with anti-tropomyosin and FITC-labeled sheep anti-rabbit IgG 143 26. Fluorescence and phase micrographs of porcine papillary myofibril after incubation with anti-a-actinin and FITC- labeled sheep anti-rabbit IgG 146 vi Figure Page 27. Fluorescence and phase micrographs of porcine papillary myofibril after incubation with anti-tropomyosin and FITC-labeled sheep anti-rabbit IgG 146 28. Electron micrographs of porcine psoas muscle showing location of anti-a-actinin and anti-tropomyosin binding 149 29. Electron micrographs of porcine papillary muscle showing location of anti-a-actinin and anti-tropomyosln binding 151 30. Electron micrographs showing binding of anti-a-actinin to an Intercalated disk in porcine papillary muscle 154 31. Electron micrographs of semiadjacent sections of porcine papillary muscle that show binding of anti-a-actinin to two regions of the intercalated disk 157 32. Electron micrographs of semiadjacent sections of porcine papillary muscle that show binding of anti-tropomyosln to different regions of the Intercalated disk 159 33. SDS-polyacrylamide gel electrophoresis of myofibrils from porcine psoas and porcine papillary muscle 161 34. Electron micrograph of glycerinated human nemaline myopathy muscle showing numerous rod bodies (KB) located near the nucleus where they are often seen in nemaline myopathy muscle 164 35. Electron micrograph of glycerinated human nemaline myopathy muscle showing fusiform expansions of the Z-disk (F) characteristic of nemaline myopathy muscle 164