Chemical and Structural Differences Between Cilia and Flagella from the Lamellibranch Mollusc, Aequipecten Irradians

R- W. Linck R. W. LINCK* Gill cilia and sperm flagella from the lamellibranch mollusc Aequipecten irradians were isolated by several methods and chemically fractionated by low-ionic-strength dialysis. These organelles differ in their forms of dynein and in the stabilities of their homologous microtubules and subsidiary structures (i.e. nexin fibres and spoke material). Inflagellamore than 80 % of the axoneme ATPase is solubilized, appearing as a 14S peak in the ultracentrifuge, and the whole axoneme is broken down to unlinked, doublet outer fibres. In cilia only lialf of the ATPase is solubilized from axonemes as a 14s component. The ciliary B-tubules and one member of the central pair also dissolve, leaving the A-tubules and the other central tubule held together by nexin fibres and matrix material as a 9 + 1 singlet axoneme, to which is bound the remaining half of the ATPase. This tightly bound form of the ciliary ATPase can be removed in an enzymically active form by brief trypsin treatment which causes the breakdown of the singlet axoneme. The trypsin-solubilized ATPase behaves like native 14s dynein in the ultracentrifuge but breaks down to polypeptides when electrophoresed in the presence of sodium dodecyl sulphate (SDS). CHEMICAL AND STRUCTURAL DIFFERENCES AND FLAGELLA MOLLUSC, AEQUIPECTEN IRRADIANS The 9 + 2 microtubular axoneme with its outer fibre arms, radial spokes and circumferential linkage fibres is a remarkably constant feature of cilia and flagella throughout the plant and animal kingdoms (Allen, 1968; Gibbons, 1961; Gibbons & Grimstone, i960; Phillips, 1970; Ringo, 1967; Warner, 1970). Although structurally similar, cilia and flagella are nevertheless functionally different (Sleigh, 1962). Cilia exert a unidirectional force on the surrounding media parallel to the cell membrane. Flagella develop a net force which is directed along the flagellar axes and away from the basal bodies. Generally speaking, movement of these organelles arises from the proximal generation of a bending region and a propagation of that bend distally (Gibbons & Gibbons, 1972; Gray, 1955; Aiello & Sleigh, 1972). Summers & Gibbons (1971) have now presented strong evidence that flagellar bending results from the sliding of adjacent outer doublet microtubules, possibly mediated by the ATPase arms. Gibbons (1963,1965,1966) has fractionated and characterized the axoneme ATPase dynein and localized its site as the 2 arms on the doublet outer fibres. From Tetrahymena cilia 2 forms of dynein were obtained having sedimentation coefficients of 14 s and 30 S (Gibbons, 1963; Raff & Blum, 1969). The 2 dyneins differ only slightly in their enzymic properties (Gibbons, 1966) and have been related as monomer and polymer (Gibbons & Rowe, 1965). Dynein has also been obtained from sperm flagella of sea urchins (Gibbons, Fronk & Gibbons, 1970; Gibbons, 1965; Mohri, Hasegawa, Yamamoto & Murakami, 1969) and from starfish (Linck, unpublished observations), but in these cases only the 14 s form has been observed. Tetrahymena cilia and sea-urchin sperm flagella also behave differently with regard to the linkages which maintain their 9-fold cylindrical symmetry (Gibbons, 1965). Stephens (19706, 1971) has isolated the linkage protein 'nexin' and has demonstrated that these linkages interconnect the outer A-subfibres. Differences in the forms of dynein and nexin have important consequences for a sliding filament mechanism and may offer an explanation for the different modes of ciliary and flagellar bending. In order to resolve the differences previously observed in unrelated species, cilia and flagella were compared from a single source, the lamellibranch mollusc Aequipecten irradians. Unless specified below, the materials and methods for this investigation are identical to those outlined previously (Linck, 1973). Fractionation of ciliary and flagellar axonemes Axonemes were suspended in Tris-EDTA solution (01 ITLM EDTA, I HIM Tris, pH 8-3 at o CC) at protein concentrations of approximately 5-10 mg/ml and dialysed against at least 100 volumes of the same solution at 0-4 C for 48-60 h. The dialysis solution was changed every 12 h. After dialysis the suspensions were separated into supernatant and pellet fractions by centrifugation at 3 5 000 g for iomin; dialysed flagella preparations required the presence of salt (10 mM Tris) for proper sedimentation. Protein and enzyme balances were then obtained by one of two procedures: (a) by direct determination from the supernatant and washed pellet fractions, or (b) by calculation from values of the supernatant fraction and a sample of the whole suspension taken prior to centrifugation. Both methods provided comparable results. In some cases o-i mM A T P , pH 7-0, was included in the dialysis solution, as it helped to preserve the enzymic activity of flagellar dynein. Adenosine triphosphatase activity ATPase activity was assayed according to the procedure described in the preceding paper (Linck, 1973). The Mg*+-activated ATPase was measured in the presence of 1 mM A T P , 30 mM Tris, 1 mM MgCl2 and o-i mM E D T A , p H 8-o, at 20 CC. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis was carried out according to the procedures of Shapiro, Vifiuela & Maizel (1967) and Weber & Osborn (1969). Pellets of protein were resuspended in SDS-medium containing 1 % SDS, 10 mM phosphate buffer, p H 7-0, 1 % /?-mercaptoethanol, 1 0 % glycerol and 0-005 % bromophenol blue indicator. Solutions or suspensions of protein were diluted 1:1 with a twice-concentrated solution of SDSmedium. Each sample was dissolved by mixing and then heating to i o o C for 2-5 min. An acrylamide stock solution was made by dissolving 30 g of acrylamide (Eastman-Kodak) and o-8 of JV-iV'-methylene-bisacrylamide in deionized water and making up to 100ml. T h e acrylamide gel concentration found most effective for these studies was 3 %. A typical set of gels, 80 mm long by 6 m m in diameter, was prepared from a mixture of 2-0 ml of T O M phosphate buffer, pH 7-0, 0-2 ml of 10 % SDS, 2-0 ml of stock acrylamide solution, 156 ml of deionized water, 10 fil of T E M E D (N,W^V',W-tetramethylethylenediamine) and 0-2 ml of 1 0 % ammonium persulphate (added immediately before pouring). After pouring, the gel3 were layered with deionized water and polymerized at 20 C. Electrophoresis was performed at a constant temperature of 20 C using a water jacket, and at a constant voltage of 50 V with an electrode buffer containing 0 1 M phosphate, pH 7-0, and o-i % SDS. Gels were normally run until the tracking dye reached the bottom, at which time they were removed and stained for 2-5 h with 0-25 % Coomassie Brilliant Blue in 5 0 % methanol containing 1 0 % acetic acid. Gels were destained by diffusion using 1 0 % methanol containing acetic acid. Mobilities (.Revalues) were calculated according to the following formula: mobility = migration distance of a given component migration of bovine serum a (...truncated)