Flagellar doublet microtubules: fractionation of minor components and alpha-tubulin from specific regions of the A-tubule

R. W. Linck 0 1 0 Laboratory of Molecular Biology, Medical Research Council , Cambridge, England CBz zQH 1 Department of Anatomy, Harvard Medical School , Boston, Massachusetts, 02115, U.S.A. (current address) FLAGELLAR AND Proteins occurring in minor amounts with purified sperm flagellar doublet microtubules were identified and studied by SDS-gel electrophoresis. Methods were developed to solubilize selectively these minor components; electron microscopy (EM) of the fractionated products revealed possible locations of these proteins in the tubule. Doublet microtubules were prepared from sea-urchin (Echinus esculentus and Strongylocentrotus droebachiensis) and scallop (Pecten maximus) sperm by dialysing flagellar axonemes against 2 mM Tris-o-2 mM E D T A - 0 5 mM D T T . E M indicates that these doublet tubule preparations retain at least 70 % of their radial spokes; cross-sections show a globule or fibre applied to the inside wall of the A-tubule, across from the inner B-tubule junction. On SDS-gels these preparations separate into at least 10 minor bands, accounting for 2 0 - 3 0 % of the total protein; the remaining 75 4 % migrates as tubulin. For E. esculentus the molecular weights and relative amounts of these components are: Component Ee 8 (150000 Daltons; 1 % ) , 11 (114000; 2-5%), 15 (89000; 2%), 16 (80000; 2-5%), 17 (74ooo; 2%), 18 (69000; 2%), 19 (66000; 2 %), 21 (48000; 4 5 %), 22 (45000; 3 %) and 23 (44500; 3 %). - Treatment of sea-urchin tubules with 0-1-0-5 % sarkosyl, 01-0-3 M KSCN or 0-3-0-6 M K I results in the selective solubilization of: first, component 8 and some B-subfibre tubulin; second, components 11 and 23 and the remaining B-subfibre tubulin; third, most of the A-subfibre tubulin and components 17, 18 and 19. Thermal fractionation extracts none of these components, suggesting they are principally associated with the A-tubule. Finally 25-35 % of the original protein is resistant to solubilization, and appears in the E M as ribbons of 3 protofilaments with 16-nm axial repeats. T h e resistant ribbons contain components 15, 16, 21 and 22 (plus component 20 in S. droebachiensis) in addition to 25 4 % of the total tubulin. T h e data support the existence of two stable moieties in each doublet tubule: (1) a ribbon of 3 protofilaments and (2) either a second ribbon of 3 protofilaments or an equivalent amount of tubulin in some other form. E M images suggest that one ribbon forms the lateral side of the A-tubule (e.g. protofilaments A i a s or A13il_, in the model) and that the globule applied to A13 may be a multisubunit complex of remaining minor components. Treatment of scallop tubules with 0-3 M KSCN preferentially extracts a-tubulin, yielding ribbons 14 protofilaments wide. T h e significance of this finding is discussed. Doublet microtubules form the outer 9 longitudinal elements of 9 + 2 axonemes in eukaryotic cilia and flagella (Fawcett & Porter, 1954; Gibbons & Grimstone, i960); they also occur in more complicated spiral arrangements in the sperm of certain insects (Phillips, 1969). The structural and perhaps chemical complexity of the doublet microtubule is evidenced by the large number of different components attached to it both circumferentially and longitudinally. One member of the doublet tubule, the A-tubule, possesses several sets of attachment sites for its accessory structures: 2 sites for the attachment of the B-tubule (Ringo, 1967; Tilney et al. 1973)> 2 fr t n e different rows of dynein arms (Afzelius, 1959; Allen, 1968; Gibbons, 1965); 2 for the nexin fibres linking the 9 tubules in a cylinder (Gibbons, 1965; Linck, 19736; Stephens, 19706, 1971); one for the attachment of the radial spokes (Afzelius, 1959; Gibbons, 1961; Hopkins, 1970; Warner, 1970); and in sperm of higher organisms, one set of attachment sites to which the outer dense fibres connect (Fawcett & Phillips, 19690,6). The doublet tubules and their associated components comprise the functional units of a cilium or flagellum, and are believed to interact by a sliding mechanism to produce and propagate bending waves (Brokaw, 1972; Gibbons & Gibbons, 1973; Satir, 1965; Warner & Satir, 1974; Summers & Gibbons, 1971, 1973). It is not yet understood how these different structures assemble asymmetrically on to the microtubule, nor how these components interact to produce bending waves. In cross-sections of sperm flagellar axonemes, which have been fixed with tannic acid, the A-tubule of the doublet appears as a cylinder of 13 protofilaments. The B-subtubule is a partial cylinder spanning a 4-5 protofilament section of the A-tubule and is composed of 10 protofilaments (Futaesaku, Mizuhira & Nakamura, 1972; Tilney et al. 1973). The 3-dimensional packing of the microtubule protein sub-units has been determined for both the A- and B-tubules (Grimstone & Klug, 1966; Amos & Klug, 1973; Linck & Amos, 1974a) by diffraction analysis of electron-microscope images of negatively stained flagellar microtubules. The cylindrical wall of the A-tubule is composed of 13 straight protofilaments 5 nm apart centre to centre, with axial repeats of 4 and 8 nm, corresponding to monomer and dimer protein subunits respectively. The individual 4-nm monomer subunits are arranged on a 3-start, left-handed helical lattice, with the 8-nm dimers of adjacent protofilaments being staggered. The surface lattice and the handedness is the same for the B-subtubule; however, instead of being staggered, dimers of adjacent protofilaments are lined up along the direction of the 3-start helix. The surface lattice of the A-tubule is essentially identical to that of singlet microtubules from brain as determined by Erickson (19746). All singlet microtubules so far tested and each subtubule of doublet microtubules are composed principally of 2 different globular protein subunits, a- and /?-tubulin, presumably associated in the form of an a/? heterodimer (Bibring & Baxandall, 1971; Bryan & Wilson, 1971; Feit, Slusarek & Shelanski, 1971; Fine, 1971; Luduena & Woodward, 1973; Olmsted, Witman, Carlson & Rosenbaum, 1971; Witman, 1970). Under denaturing conditions, both a- and /9-tubulin monomers have molecular weights of 54000 Daltons (Bryan, 1972; Lee, Frigon & Timasheff, 1973). Behnke & Forer (1967) originally demonstrated several classes of microtubules, based on relative stabilities to different treatments; in particular, the B-subtubule was found to be substantially more labile than the A. Stephens (1970a) subsequently developed procedures for thermally fractionating flagellar A- and B-tubules and found chemical differences between the major protein components of these 2 tubules. Furthermore, other investigators have fractionated doublet tubules with sarkosyl detergent and found a resistant ribbon of 3 protofilaments, which they interpret to be the 'partition', i.e. the tubule wall between the A- and B-tubules (Meza, Huang & Previous studies on the chemical compositions of purified doublet microtubules hav (...truncated)