Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1967 Formation and structure of filamentous systems for insect flight and mitotic movements Melvyn Dennis Goode Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons Recommended Citation Goode, Melvyn Dennis, "Formation and structure of filamentous systems for insect flight and mitotic movements " (1967). Retrospective Theses and Dissertations. 3391. https://lib.dr.iastate.edu/rtd/3391 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]. FORMATION AND STRUCTURE OF FILAMENTOUS SYSTEMS FOR INSECT FLIGHT AND MITOTIC MOVEMENTS by Melvyn Dennis Goode A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Subject: Cell Biology Approved: Signature was redacted for privacy. In Charge oi Major Work Signature was redacted for privacy. Chairman Advisory Committee Cell Biology Program Signature was redacted for privacy. Signature was redacted for privacy. Iowa State University Of Science and Technology Ames, Iowa: 1967 ii TABLE OF CONTENTS Page I. INTRODUCTION 1 PART ONE. THE MITOTIC APPARATUS OF A GIANT AMEBA 5 II. THE STRUCTURE AND PROPERTIES OF THE MITOTIC APPARATUS 6 A. Introduction .6 1. Early studies of mitosis 6 2. The mitotic spindle in living cells 7 3. Electron microscope studies of the mitotic apparatus 8 4. Physical structure of the isolated mitotic apparatus 11 5. Chemical structure of the isolated mitotic apparatus 13 6. Solubility properties of the isolated mitotic apparatus 15 7. Induction of "contraction" or elongation 17 B. Methods 18 1. Cells fixed during mitosis 18 2. Isolation and study of the mitotic apparatus 20 C. Results 22 1. Chemical structure of the mitotis apparatus 22 2. Fine structure of the mitotic apparatus 25 3. Structure of the isolated ameba mitotic apparatus 28 4. Solubility properties of the isolated mitotic apparatus 31 5. Experimental alteration of the isolated mitotic apparatus 37 D. Discussion 41 1. Chemical structure of the mitotic apparatus 41 2. Solubility properties of the isolated mitotic apparatus 41 3. Induction of elongation 45 E. Summary 46 III. THE MOLECULAR STRUCTURE AND FORMATION OF MICROTUBULES IN THE MITOTIC APPARATUS 48 A. Introduction 48 1. Molecular subunits of the isolated mitotic apparatus 48 iii 2. Microtubule ultrastructure 52 3. Formation of the mitotic apparatus 53 4. Experimental manipulation of microtubule assembly 59 B. Methods 60 1. Negative contracting of microtubules 60 2. Mitotic apparatus formation 61 C. Results 62 1. Microtubule ultrastructure 62 2. Mitotic apparatus formation 70 D. Discussion 76 1. Microtubule ultrastructure 76 2. Formation of the mitotic apparatus 85 3. Cellular changes during mitosis affecting mitotic apparatus formation 94 E. Summary 96 PART TWO. FLIGHT MUSCLE OF DROSOPHILA MELANOGASTER 98 IV. THE STRUCTURE AND PROPERTIES OF ADULT FLIGHT MUSCLE 99 A. Introduction 99 1. The structure of vertebrate skeletal muscle 100 2. The structure of insect flight muscle 101 3. The properties of isolated flight-muscle myofibrils 107 B. Methods 110 1. Electron microscopy of flight muscle 110 2. Studies of isolated myofibrils 111 C. Results 112 1. Fine structure of flight muscle 112 2. Properties of isolated flight-muscle myofibrils 123 D. Discussion 130 1. Fine structure of flight muscle 130 2. Properties of isolated flight-muscle myofibrils 133 V. THE MOLECULAR STRUCTURE AND FORMATION OF FLIGHT MUSCLE 138 A. Introduction 138 iv 1. The molecular subunits of muscle 138 2. The ultrastructure of myofilaments 142 3. The development of muscle 144 B. Methods 149 1. Isolation and electron microscopic study of myofilaments 149 2. Electron microscope study of flight-muscle forma­ tion 150 C. Results 150 1. Ultrastructure of myofilaments 150 2. Formation of flight muscle 156 D. Discussion - 164 1. Ultrastructure of nyofilaments 164 2. Myofilament assembly ' 170 3. Myofibril formation 172 E. Summary 174 VI. SUMMARY AND CONCLUSIONS 175 A. Summary 175 B. Conclusions 179 1. Filament structure 179 2. Solubility properties 180 3. Formation of filaments 181 4. Formation of movement systems from filaments 182 5. Induction and control of movements 183 VII. LITERATURE CITED 184 VIII. ACKNOWLEDGMENTS ' 202 1 I. INTRODUCTION A fundamental principle of biology is that cells obey the laws of chemistry and physics. From this viewpoint motion or changes in motion within living cells are considered to be the result of an unbalanced force acting on a body. Katchalsky (1963) stated that in order to convert chem­ ical energy (a scalar quantity) directly into contractile mechanical force (a vectoral quantity), structural anisotropy is required. In other wordsJ the molecules of biological movement systems producing this type of force cannot be randomly arranged but must be organized in such a manner that the properties of the structure vary in different directions. This anisotropic organization is often observed in biological move­ ment systems on two levels: it is seen as optical anisotropy or bire­ fringence with the polarizing, light microscope and as an array of parallel filaments^ with the electron microscope. Studies of changes in birefringence and fine structure indicate that birefringence can be __ the direct result of the presence of filaments (Kane and Forer, 1965; Rebhun and Sander, 1967). However, the nature of the mechanisms pro­ ducing movement and the relationship of filaments to these movements remain unresolved. Intracellular, filamentous movement systems include muscle fibrils, the mitotic apparatus (MA), cilia, flagella, axopodia, myonemes, and ^The word "filament" is used here as a general term for intracellular structures with diameters from 4-40 my and lengths much greater than the diameter. These filaments may appear solid or tubular in cross section, and are usually straight. 2 several others. Other structures such as "plasma filaments" (Komnick and Wohlfarth-Bo11ermann, 1965) and cytoplasmic microtubules (see Porter, 1966) have been related to intracellular movements because of their loca­ tion and alignment in the direction of cytoplasmic streaming or ameboid movement. But to demonstrate that these anisotropic filament arrange­ ments produce movement, rather than result from a movement gradient such as occurs in flow birefringence, seems to be a difficult problem. Nevertheless, the concept that where movement takes place, filaments are present is becoming increasingly well established, although thà con­ verse statement that movement occurs whenever filaments are present is definitely not always true (Roth et al., 1966). An attractive unifying hypothesis is that most types of cellular movement, including mitotic movements and the contraction and relaxation of muscle, are modifications of a common molecular mechanism. For ex­ ample, Heidenhain (1895) and Weber (1958) both stressed the analogy of muscle and the MA. on the basis of physiological properties. This hypoth­ esis is supported by the experiments of Hoffmann-Berling (1954), who found that glycerine-extracted, anaphase-cell models can be induced to "contract" or elongate under suitable conditions of ATP concentration, ionic strength, etc., in a manner similar to the response of muscle-fiber models prepared in the same way. However, he could not demonstrate whether these changes were a response of the MA or the elongation of the entire cell. Immunochemical reactions indicate that the analogies between the two systems should not be extended to imply that genetically similar molecules are utilized. Fluorescent antibodies to chick muscle proteins are not selectively localized in dividing chick fibroblasts (Holtzer 3 et al., 1959), and no immunochemical reaction between sea-urchin muscle extracts and MA-protein antiserum is observed (Went and Mazia, 1959). Although no myosin-like protein is found in the MA (Inouë, 1964), similarities between MA protein and actin are shown by their reactions with myosin. Protein extracted from the isolated MA with a method used to extract actin from muscle will react with rabbit-muscle myosin A to form a superprecipitate similar to the vitro actin-myosin inter­ action (Miki and Oosawa, 1962). This extract also activates the myosin- ATPase activity in the presence of magnesium. An actin-like protein with similar properties can be isolated from calf-thymus nuclei (Ohnishi et al., 1964). In addition fluorescein-conjugated heavy meromyosin reacts with the spindle fibers of the isolated sea-urchin MA as well as with the actin-containing I bands of isolated myofibrils (Aronson, 1965). Thus, some experimental basis does exist for a comparison of the proper­ ties of the MA and muscle. The intent of this dissertation is to characterize broadly the properties of two filamentous movement systems, the mitotic apparatus and flight-muscle myofibrils, and to form some comparative conclusions on the basis of similar studies on each system. In each case information was sought to clarify the molecular architecture of the filaments in­ volved, the bonds or forces that may maintain or alter the structure of the systems, the mode of