Comparative Isolation of Cilia and Flagella from the Lamellibranch Mollusc, Aequipecten irradians

R. W. LINCK 0 0 The Department of Biology, Brandeis University , Waltham , Massachusetts 02154 and the Marine Biological Laboratory , Woods Hole, Massachusetts 02543 , U.S.A SUMMARY Gill cilia and sperm flagella from the lamellibranch mollusc Aequipecten irradians were compared with respect to their ultrastructures and adenosinetriphosphatase activities. Cilia were isolated from excised gills using 3 different solutions: twice-concentrated seawater, 10% ethanol-10 mM CaCl, and 60% glycerol. In each case deciliation occurs by the severance of the cilium at the junction of the transition zone and the basal body, and in each case the ciliary ultrastructure is maintained. Sperm flagella were purified by mechanical decapitation. Cilia and sperm flagella have similar fine structures, except that the matrix of the cilia contains substantially more electron-dense material than that offlagella.The ATPase activity of purified cilia is approximately 009 fimol P^min/mg protein; that offlagellais 0-13. Ciliary and flagellar axonemes were prepared by repeated extraction of the membranes with 1 % Triton X-100. Ciliary axonemes maintain their 9 + 2 cylindrical orientation, whereas flagellar axonemes often appear as opened or fragmented arrays of the 9 + 2 structure, due to the partial breakdown of the flagellar nexin fibres. A-subfibre arms which were obvious in whole organelles are rarely seen in axoneme preparations. Again the ciliary matrix is considerably more amorphous than in flagellar axonemes. The ATPase activities of ciliary and flagellar axonemes are 013 and o-12/imol P|/min/mg protein respectively; however, activities of ciliary axonemes may vary by a factor of 2, depending on the method of isolation. The difficulty in observing A-subfibre arms in cross-sections of ciliary andflagellaraxonemes is discussed in terms of random, nonreinforcing arrangements of the dynein arms. - A number of investigators have reported methods for the isolation of cilia and flagella and have characterized the major components of these organelles (Gibbons, 1968; Stephens, 1971), but a comparison of cilia and flagella from the same species has not been reported. Considering that cilia and flagella differ in their modes of beat (Gray, 1928, 1955; Sleigh, 1962), such intraspecies comparisons might reveal structural or biochemical differences relevant to the phenomenon of wave propagation. Since lamellibranch molluscs provide a constant source of gill cilia and an adequate, seasonal supply of sperm (Sastry, 1970), the bay scallop Aequipecten irradians was investigated. Three previous methods of cilia isolation were modified for harvesting 3 isolation methods were compared on the basis of the yields, the ATPase activities and the ultrastructure of whole cilia and Triton-extracted ciliary axonemes. Flagella and flagellar axonemes were obtained from ripe scallops and similarly investigated. This report deals with the physiological and structural differences between the isolated organelles. Ciliary and flagellar axonemes from A. irradians were then chemically fractionated and found to differ in their forms of dynein and in the stabilities of their homologous microtubules and secondary structures; these results are presented in the following paper (Linck, 1973). MATERIALS AND METHODS Solutions, reagents and conditions Solutions frequently used in this report have been abbreviated as follows: Tris-Mg solution denotes 30 mM Tris (tris(hydroxymethyl)aminomethane), 3 mM MgClj, o-i IDM EDTA (ethylenediaminetetra-acetate), pH 8 3 at o C; Tris-Mg-Triton solution, 30 mM Tris, 3 mM MgCl2) 0 1 mM EDTA, 1% Triton X-100, pH 83, at o C; and EDTA-seawater, filtered seawater made 0 1 mM in EDTA. Adenosine triphosphate, obtained from Calbiochem and P-L Biochemicals, was stored as the powder or as a neutralized 01 M stock solution in a 20 C deep freeze. All procedures were carried out at o C unless otherwise specified. The cheese cloth used in the cilia and sperm tail isolation procedures was first soaked in EDTA-seawater. Procedures for the isolation of gill cilia Lamellibranch molluscs of the species Aequipecten irradians (formerly Pecten irradians) were obtained from the Marine Biological Laboratory, Woods Hole, Massachusetts. The gills were excised, collected in roughly 10 times their volume of cold EDTA-seawater and washed twice more by transfer to fresh EDTA-seawater to remove silt and mucous. In some cases the wet weight of the gills was recorded in order to calculate yields. Three previous methods of cilia isolation were modified for harvesting gill cilia: (1) The 2 x seawater procedure (Auclair & Siegel, 1966; Stephens & Linck, 1969). Gills were suspended in roughly 10 times their volume of twice-concentrated EDTA-seawater (30 g of NaCl per 1. of EDTA-seawater) at room temperature and stirred gently but continuously for 10 min, at which point a maximally cloudy suspension of cilia and fragmented gills was obtained. It was noted that in this procedure the gills do not deciliate as readily in cold hypertonic seawater. The suspension was filtered through cheese cloth and then centrifuged at 1500 g for 5 min in 50-ml tubes or for 10 min in 250- or 300-ml bottles. The supernatants were carefully collected and recentrifuged at 10 000 g for the same length of time to pellet the essentially pure cilia. (2) The ethanolcalcium procedure (Watson & Hopkins, 1962; Gibbons, 1965 a). Each 10 g of gills was suspended in 100 ml of medium consisting of 10% ethanol, 0 1 M NaCl, 2 mM EDTA, and 30 mM Tris, pH 80, at room temperature. The gills were stirred and allowed to equilibrate for 2 min, after which the suspension was made 10 mM in CaCls by rapidly adding from a 1 M stock solution and stirring continuously. At room temperature the deciliation began immediately and reached a maximum in about 4 min (about twice as long at o C). The suspension was filtered and centrifuged as above to obtain a pellet of cilia. (3) The glycerol procedure (Gibbons, 1965 b). Approximately 20 g of wet gills were suspended in 200 ml of 70% glycerol, 30 mM KC1, 25 mM MgCl2, o-i mM EDTA, and 30 mM Tris, pH 80, at room temperature, and stirred continuously for 15 min. After filtering through a thin layer of cheese cloth, the suspension was cooled to o C and centrifuged at 12000 g for 10 min using half-full 50-ml tubes. The supernatant was carefully collected and centrifuged at 80000 g for 3 h to pellet the cilia. The pellets of cilia obtained from a given procedure were consolidated and washed once with 20 vol. of Tris-Mg solution for use as whole cilia, or washed 3 times with 20 vol. of TrisMgTriton solution, followed by 1 wash with Tris-Mg solution, for use as ciliary axonemes. Three washes of the Tris-Mg-Triton solution were found to remove essentially all of the ciliary membranes. T h e washed cilia or ciliary axonemes were collected by centrifugation at 10000 g for 5 and 10 min, respectively. Cilia preparations were routinely examined und (...truncated)