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An Actin-Like Component in Sperm Tails of a Crane Fly (Nephrotoma Suturalis Loew)

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An Actin-Like Component in Sperm Tails of a Crane Fly (Nephrotoma Suturalis Loew) J. Cell Sci. II, 491-519 (1972) 491 Printed in Great Britain AN ACTIN-LIKE COMPONENT IN SPERM TAILS OF A CRANE FLY (NEPHROTOMA SUTURALIS LOEW) A. FORER* Molecular Biology Institute, Odense University, Niels Bohrs Alle, DK-5000 Odense, Denmark AND O. BEHNKE Anatomy Institute C, Copenhagen University, Raadmandsgade 71, DK-2200 Copenhagen N, Denmark SUMMARY In negatively stained preparations made from glycerinated crane fly spermatids and sperm, actin-like filaments are seen which bind heavy meromyosin (HMM) to form arrowhead complexes, the reaction with HMM being blocked by ATP and pyrophosphate. In preparations from young spermatids the actin-like filaments are found singly, or in small groups, while in those from mature sperm the actin-like filaments are organized into a structure which we call 'rods'. Both rods and single filaments come from lysed sperm tails. The actin-like filaments in rods bind HMM only when frayed out on a grid. In sections of normal or glycerinated spermatids or sperm, no actin-like filaments are seen, either because they are not preserved through the fixation and embedding procedures, or be- cause they are present in a form which we do not recognize. In sections of glycerinated sperm- atids incubated with HMM, decorated filaments are seen in non-nuclear regions of spermatids (tails), oriented parallel to the axoneme. These probably correspond to the single filaments identified in the negatively stained preparations and not to the rods, because the actin-like filaments in rods bind HMM only after fraying out on a grid. HMM causes polymerization of filaments: actin-like filaments are not seen in negatively stained preparations of glycerinated cells subsequently incubated in salts solution, yet are seen in such cells after a further incubation in HMM. Thus some of the decorated filaments seen in sections may have been polymerized by the HMM, raising questions about the procedure. After rupture of living cells no single actin-like filaments are seen in negatively stained preparations, raising the question of whether glycerol as well as HMM can cause polymeriza- tion of filaments. Rods are seen in such preparations, and thus rods seem to exist as such in living cells. Single actin-like filaments and rods are seen even when cytoplasmic microtubules are not seen (after incubation in colchicine or vinblastine). Thus the filaments do not seem to come directly from cytoplasmic microtubules. The possible presence of actin-like filaments in cilia and in other flagella, the location of actin-like filaments in sperm tails, and the possible role of the actin-like filaments are discussed. INTRODUCTION F-actin can be identified in preparations negatively stained and observed in an electron microscope: F-actin has a characteristic morphology (Hanson & Lowy, 1963), and binds heavy meromyosin (HMM) to form arrowhead complexes (Huxley, 1963). * Present address: Biology Department, York University; 4700 Keele Street, Downsview 463, Ontario, Canada. 492 A. Forer and O. Behnke Thin (5-6 nm) filaments found in a variety of cells have been identified as 'actin-like' (or 'actinoid') because these filaments become decorated with arrowheads after the cells are glycerinated and HMM is added to the external medium (for example, see Ishikawa, Bischoff & Holtzer, 1969; Pollard & Korn, 1971; Perry, John & Thomas, 1971; Behnke, Kristensen & Nielsen, 1971a, b; Allison, Davies & de Petris, 1971), and because ATP or pyrophosphate prevents the HMM binding (Huxley, 1963; Ishikawa et al. 1969; Behnke et al. 19716). Actin filaments are associated with muscle contrac- tion (Huxley, 1969). Since actin-like filaments seem to be associated with amoeboid movements (Pollard & Ito, 1970; Pollard & Korn, 1971; Nachmias, Huxley & Kessler, 1970; Wooley, 1970) and are found in many different kinds of cells, from protozoa (above) to those of man (Behnke et al. 1971a, b), and could be responsible for the motility in these cells (in conjunction with corresponding myosin-like filaments), it seemed likely that all motile systems contain actin-like and myosin-like proteins which interact to cause motility. We therefore looked in sperm tails for actin-like filaments (i.e. those giving arrowhead-complexes with HMM). In this report we describe the occurrence, localization and some properties of actin-like filaments found in sperm tails of crane flies. METHODS Animals and testes Crane flies {Nephrotoma suturalis Loew) were reared in the laboratory (Forer, 1964). All the cells in any one crane fly testis are spermiogenic and are in roughly the same stage of develop- ment at any given time. We studied the following stages of maturation of the sperm: (1) 'young spermatids', in testes which contained both spermatocytes and spermatids, about 8-9 days after the fourth larval instar; (2) 'middle larva spermatids', in testes which contained only spermatids, about 1 day after meiosis; (3) 'old larva spermatids', in testes dissected from larvae about 1 day before pupation and about 2 days older than the 'middle spermatid' larvae; (4) 'pupa spermatids', in testes dissected from pupae, about 1-2 days after pupation; and (5) 'mature sperm', in testes dissected from virgin adult males. Only mature sperm are motile. Dissections were performed under oil, as described elsewhere (Forer, 1965), and testes remained under oil until transferred to one of the experimental solutions. Solutions The compositions of the various solutions are given here: the exact sequence in which the solutions were used will be described in the text, as will temperatures. For all glycerinations, the glycerol solution was at room temperature. 'Glycerol I standard salts' (G/SS) is a 1:1 mixture of pure glycerol plus standard salts solution. ''Glycerol'/high salts' (G/HS) is a 1:1 mixture of pure glycerol plus high salts solution. Standard salts solution (SS) contained (final concentrations) 0-05 M KC1, 0005 M MgCl2, and 0006 M Sorensen's phosphate buffer (KH2PO4/Na2HPO4), and had a final pH of 70. High Salts solution (HS) contained (final concentrations) 0-5 M KC1, 0-005 M MgCL, and 0006 M Sorensen's phosphate buffer, at a final pH of 7-0. 'ATP/standard salts' (ATP/SS) contained 0-008 M disodium adenosine-s'-triphosphate (Sigma) in SS, at a final pH of 67. 'Pyrophosphatejstandard salts' contained 001 M sodium pyrophosphate in SS, at a final pH of 6-8. Ringer's solution was essentially Ephrussi-Beadle insect Ringer solution (Ephrussi & Beadle, 1936) with buffer, and contained (final concentrations) 013 M NaCl, 0005 M KC1, 0003 M CaClj, and 001 M Na,HPO</NaH£PO4 buffer, at final pH 6-8. Colchicine solution contained colchicine in Ringer at a final concentration of io~3 M. Vinblastine sulphate ('Velbe', Eli Lilly) was at a final concentration of io~3 M in Ringer. Actin-like component in sperm tails 493 After testes were placed in colchicine solution they were either fixed with glutaraldehyde or transferred to other solutions: all subsequent solutions (until fixation or lysis for negative staining) contained io~3 M colchicine. HMM (heavy meromyosin) was prepared as described previously (Behnke & Emmersen, 1972), and stored at — 13 CC after mixing 1:1 with glycerol/ SS; this stock HMM had 10 mg of HMM per ml. When testes were placed in HMM solution prior to embedding for electron microscopy the HMM solution contained 1-2 mg of HMM per ml, the stock HMM being diluted with the solution in question. When used for negatively stained preparations, the HMM solution was diluted with SS to give a final concentration of 0-2-0-4 mg of HMM per ml. Before each negative staining experiment utilizing HMM, a preparation was made of HMM solution alone, and carefully checked for the absence of decorated filaments. Fixation and embedding Testes were fixed with 2 % glutaraldehyde solution which was at the same temperature as the testes (either 4 °C or room temperature). The glutaraldehyde was either 2 % in the same solution as were the testes immediately prior to fixation, or 2% in o-i M cacodylate buffer, pH 68. The testes were placed in glutaraldehyde for between 2 and 36 h, rinsed in SS, post- fixed for 2 h in 1 % osmium tetroxide (in SS), rinsed in H2O, and left overnight in 1 % uranyl acetate (in H2O). They were then dehydrated through a graded series of ethanols, followed by propylene oxide, and embedded in either Araldite or Epon. Sections were stained with 1 % uranyl acetate (in H2O), followed by lead citrate (Reynolds, 1963), and were observed using a Philips EM 300 electron microscope. Negative staining Testes were placed in a drop of solution (described in the text), the testes sheaths were removed, and the contents of each testis sucked into a i-ml syringe fitted with a 26 G gauge hypodermic needle, and the cells ruptured by repeatedly and forcibly expelling and sucking the drop through the syringe. Drops were then placed on carbon-Formvar coated grids and after about 1 min, blotted off. HMM was added at this point for some grids, and after 1 min was blotted off. The grids (either with or without HMM) were then rinsed with a few drops of 1 % formalin (in SS), fixed for 1 min with 1 % formalin, and negatively stained with 1 % uranyl acetate (Huxley, 1963). RESULTS Actin-like filaments in negatively stained preparations Specificity of HMM-binding. Actin-like filaments were identified in negatively stained preparations (and in sections) by their ability to form arrowhead complexes with HMM. While there is no evidence indicating that the reaction is not specific, several workers have discussed the question of specificity and have shown that, as observed in negatively stained preparations, HMM does not bind to collagen fibrils or 10-nm diameter filaments (Ishikawa et al. 1969; Hitchcock, 1971), and, as observed in sections, HMM does not bind to 'tonofilaments', to collagen, or to intact micro- tubules (Ishikawa et al.
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