Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1991 Selected properties of the cytoskeletal protein synemin and its interactions with proteins at the muscle Z-line Stephan Roman Bilak Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, and the Cell Biology Commons Recommended Citation Bilak, Stephan Roman, "Selected properties of the cytoskeletal protein synemin and its interactions with proteins at the muscle Z-line " (1991). Retrospective Theses and Dissertations. 9629. https://lib.dr.iastate.edu/rtd/9629 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]. UMI MICROFILMED 1992 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly fi"om the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is atso photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9212181 Selected properties of the cytoskeletal protein synemin and its interactions with proteins at the muscle Z-line BilaJc, Stephan Roman, Ph.D. Iowa State University, 1991 UMI 300N.ZeebRd. Ann Aibor, MI 48106 Selected properties of the cytoskeletal protein synemin and its interactions with proteins at the muscle Z-line by Stephan Roman Bilak A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Animal Science Major: Muscle Biology Approved: Members of the Committee: Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. Signature was redacted for privacy. theMajor Depé^ent Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1991 ii TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 SECTION I. SELECTED PROPERTIES OF MUSCLE SYNEMIN AND IDENTIFICATION OF A MAMMALIAN HOMOLOG 39 SUMMARY 41 INTRODUCTION 43 MATERIALS AND METHODS 46 RESULTS 58 DISCUSSION 103 ACKNOWLEDGMENTS 113 REFERENCES 114 SECTION II. INTERACTION OF PURIFIED SYNEMIN WITH MUSCLE Z-LINE PROTEINS 121 ABSTRACT 123 INTRODUCTION 125 MATERIALS AND METHODS 128 RESULTS 136 DISCUSSION 151 ACKNOWLEDGMENTS 157 REFERENCES 158 ill OVERALL SUMMARY 165 LITERATURE CITED 169 ACKNOWLEDGMENTS 195 1 GENERAL INTRODUCTION Vertebrate Skeletal Muscle Structure and Major Properties of the Myofibrillar Proteins Tliree types of muscle are recognized in vertebrates, including skeletal (striated and voluntary), cardiac (striated and involuntary), and smooth (nonstriated and involuntary) muscles. The fundamental structural unit of skeletal muscle is the individual muscle fiber or cell [for reviews, see Goll et al., 1984; Huxley, 1988]. The muscle cell cytoplasm (sarcoplasm) of the multinucleated fiber contains the usual insoluble complement of subcellular constituents such as the Golgi apparatus, mitochondria, ribosomes, glycogen granules, and endoplasmic (sarcoplasmic) reticulum. When the muscle cell is viewed with the light microscope, alternating dark (A-band) and light (l-band) bands are observed [Huxley, 1953, 1957, 1988; Huxley and Nigerderke, 1954]. This banding pattern is the result of the presence of a highly organized array of cylindrical protein threads (1-2 //m in diameter), called myofibrils, which are oriented in register and parallel to each other and which extend the length of the muscle fiber. Electron microscopy conducted on thin sections of striated muscle reveal that the A-band and l-band pattern results from two interdigitating filamentous systems, namely the thick (myosin-containing) and thin (actin-containing) filaments, which alternate and produce the characteristic striations observed along the myofibril [Huxley, 1957, 1963, 1988]. The H-zone is a less electron- dense region in the center of the A band of relaxed myofibrils where thin filaments are absent. A narrow, electron-dense M-line bisects the A-band [Knappeis and Carlsen, 1968; Luther and Squire, 1978]. Each i-band is bisected by a very electron-dense Z-line structure. The sarcomere is considered the basic structural unit of the myofibril and the physiological unit of muscular contraction, and is defined by the distance (-2.3-2.8 //m in resting muscle) from one Z-line to the next Z-line. The thin filaments are anchored at the Z-line at one end, where they form a square lattice in cross section, and are present throughout the I-band. The free ends of the thin filaments extend some distance (to the edge of the H-zone) into the A-band, where they are arranged in a hexagonal array in cross section [Huxley, 1957]. Thick filaments (~15 nm in diameter) are composed primarily of myosin, which is a long (-160 nm), mostly rod-shaped (-150 nm) molecule with double "heads" or globular projections (-15-20 nm long) at one end [for a review, see Squire, 1986]. Myosin is a hexamer (three pairs of subunits), containing two major (heavy) polypeptide chains (-200 kD each) and four light chains (-20 kD each). The heavy chains form all of the long myosin rod (a coiled-coil structure) and most of the myosin head region. One each of two pairs of light chains bind to each of the globular heads [Kendrick-Jones et al., 1976; Squire, 1986]. Two important properties of myosin are that it is an adenosine triphosphatase (ATPase), and that it interacts with actin during muscle contraction [Tokunaga et al., 1987]. Proteolytic fragmentation studies have shown that each myosin head region contains both ATPase activity and the actin-binding site [Vibert and Cohen, 1988]. Myosin molecules will spontaneously assemble into thick filaments in vitro, first by packing in a tail to tail (antiparallel) fashion and, secondly, in a head to tail (parallel) fashion as the filament elongates [Offer, 1974]. Presumably, this process occurs in vivo, in such a way that the rod portions of myosin molecules form the cylindrical backbone of the thick filament and the molecules in one half of a thick filament have the opposite orientation (polarity) to those in the other half. As a result, the central region of each thick filament (bare zone or pseudo H-zone) is free of the myosin head projections, which project from the thick filament surface at defined, ~43 nm intervals [Huxley, 1963; Offer, 1974; Squire, 1986]. C-protein (~140 kD) [Offer et al., 1973; Starr and Offer, 1978], initially identified in myosin preparations by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [Starr and Offer, 1971], has been shown to bind to myosin. In the muscle thick filament, C-protein is found in the cross bridge region in each half of the bipolar thick filament in seven to nine evenly-spaced bands (43 nm apart) [Offer et al., 1973]. The physiological role of C-protein remains unknown, although it has been suggested that it may function to stabilize thick filament structure during contraction and/or have a role in control of thick filament assembly [Offer et al., 1973; Starr and Offer, 1978; Koretz, 1979]. It also has been suggested [Yamamoto and Moos, 1983] that C-protein 4 may play a role in contraction because it affects actomyosin ATPase activity [Offer et al., 1973] and can bind directly to F-actin [Moos et al., 1978]. Recent, primary structural studies (from cDNA derived sequence) have shown that C-protein belongs to the immunoglobulin superfamily [Einheber and Fischman, 1990]. Thick filaments are held in register at their centers by M-line structures [Knappeis and Carlsen, 1968; Carlsson and Thornell, 1987]. Three to five sets (when viewed longitudinally; depending on the muscle fiber type and/or species) [Pepe, 1983] of transverse M-bridges connect each thick filament in a hexagonal arrangement to six neighboring thick filaments, which can be seen in cross section through the M-line region [Knappeis and Carlsen, 1968; Trinick and Lowey, 1977]. Thin M-filaments, running parallel to the myofibril axis, are thought to interconnect the transverse M-bridges at their midpoints [Luther and Squire, 1978]. In addition, Y-shaped secondary M-bridges are thought to connect adjacent M-filaments in transverse register [Luther and Squire, 1978; Carlsson and Thornell, 1987]. Myomesin (-185 kD) [Grove et al., 1984, 1985] and muscle specific creatine kinase (~84 kD, consisting of two ~42 kD homodimers) [Turner et al., 1973; Wallimann et al., 1983] are thought to make up at least some of the M-bridges. Another protein considered an M-line constituent is M-protein (~165 kD) [Trinick and Lowey, 1977], and it may comprise the M-filaments [Woodhead and Lowey, 1982].