Structure and chemical composition of insoluble filamentous components of sperm flagellar microtubules

0 Department of Anatomy, Harvard Medical School , 25 Sfull tuck Street, Boston, Massachusetts 02115 , U.S.A OF INSOLUBLE OF SPERM SUMMARY By progressive solvent extraction, we have obtained a series of subfragments of flagellar microtubules. Mild treatment gives rise to ribbons that contain longitudinally arranged protofilaments. Further extraction leaves a distinctive residue containing thinner ribbons, of three and eventually two protofilaments. Finally, filaments 2-3 nm in diameter and fibrous ribbons apparently containing 6 or more 2 nm subfibrils are found. This latter solvent-resistant material is consistently enriched in a characteristic set of polypeptides, which are found in flagella of several different species, including echinoderms and a mollusc. These polypeptides appear different from a- and /9-tubulin on the basis of their solubilities, isoelectric points and electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels; these conclusions are reinforced by peptide mapping after limited proteolytic digestion, although the latter method reveals certain similarities between these unique flagellar proteins, tubulin, chicken gizzard desmin and rabbit actin. A remarkable feature of the protein in the final fraction is the high a-helical content: 71 % as measured by circular dichroism. We consider the possible origins of these filaments in the microtubule, in particular the possibility that microtubule protofilaments are heterogeneous in protein composition, and we discuss some of the implications of our findings. - Flagellar microtubules are uniquely suited for the study of microtubule structure and chemistry, since they can be isolated in their native state and free of contamination from other cellular elements (Gibbons, 1965; Stephens & Edds, 1976). It has previously been shown that flagellar doublet microtubules can be fractionated into chemically resistant ribbons of protofilaments, suggesting microheterogeneity within the walls of microtubules (Behnke & Forer, 1967; Linck, 1976; Meza, Huang & Witman, Carlson & Rosenbaum, 19726). Our own studies suggested a preliminary explanation for the stability of these protofilament ribbons; i.e. the ribbon fraction, when compared to doublet microtubules, was found to be composed of the classical a- and /?-tubulins, together with a unique set of polypeptides specifically associated with the ribbon moiety (Linck, 1976; Linck & Langevin, 1981). It was suggested at the time that these unique proteins might account for the stability of the protofilament More recently we have studied the arrangement of subunits in the walls of flagellar microtubules (Linck & Langevin, 1981; Linck, Olson & Langevin, 1981; Woodrum & Linck, 1980). Our analyses indicated that structurally or chemically unique 'seams' may exist between certain protofilaments in native central-singlet microtubules and suggested that singlet microtubules might also possess stable protofilament ribbons in their walls. We report here the results of further investigations on the stable protofilament ribbons isolated from flagellar axonemes and discuss the possible interpretations and implications of our results. Preliminary reports of this work have been published elsewhere (Linck et al. 1981; Linck, 1982; Linck, Albertini, Kenney & Langevin, 1982). MATERIALS AND METHODS T h e sperm flagellar microtubules used in this study were from sea urchins (Strongylocentrotus droebachiewis and purpuratus) and clams (Spisula tolidissima). Axonemes and doublet tubules were purified as previously described (Linck & Langevin, 1981); they were fractionated with varying concentrations of NaSCN or urea and analysed according to the quantitative pelletassay procedure of Linck (1976). For controls, axonemes were resuspended in a final concentration of 0-15 M-KC1, 10 mM-Tris, 5 mM-MgClj, 0-5 mM-ethylenediaminetetra-acetate (EDTA), 1 mM-ATP, 1 mM-dithiothreitol ( D T T ) (pH 8-3), and doublet tubules in a final concentration of 0-15 M-KC1, 10 mM-Tris, o-i mM-EDTA, 1 mM-DTT, p H 8-3. Fractionated samples were resuspended to a final protein concentration identical to the controls, in 10 mM-Tris, 1 mME D T A , 1 m M - D T T (pH 8 3 ) and varying concentrations of NaSCN or urea. Samples were extracted for 0-5 h and centrifuged at 100000 # for 90 min. The supernatants were discarded and the pellets freeze-dried. Duplicate fractions were prepared in parallel for negative-stain electron microscopy (EM). Flagellar B(a/?)-tubulin was purified from S. purpuratus by thermal fraclionation (Stephens, 1970) and subsequent polymerization in vitro as previously described (Linck & Langevin, 1981). Desmin was purified from chicken gizzards by the procedure of Geisler& Weber (1980); the final preparation contained filaments 10-12 nm in diameter, as judged by negative-stain EM, and was composed principally of 55000 M, polypeptides. Actin was purified from rabbit skeletal muscle (see Wilson, 1982). Bovine serum albumin was purchased from Sigma Chemical Company. Sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis (PAGE) was carried out according to the procedure of Laemmli (1970) with a o-6 mm thick slab gel apparatus. For analysis of flagellar microtubules the freeze-dried pellets described above were dissolved in identical volumes of SDS sample buffer, boiled for 2 min and then dialysed against the same for 12-18 h. Identical sample volumes were applied to each lane. Electrophoresis was carried out at a constant 100 V, until the dye front moved through a 10 mm 3 % stacking gel and a 130 mm 7 % running gel. A series of protein standards were electrophoresed for molecular weight estimations (Linck & Langevin, 1981). Gels were stained in 0-025 % Coomassie Brilliant Blue R in 25 % isopropanol/10 % acetic acid and destained in 10 % acetic acid. Two-dimensional electrophoresis was performed according to O'Farrell (1975), with some modifications. T h e isoelectric focusing (IEF) gel was composed of 4-0 % acrylamide, 0-2 % bisacrylamide, i - 6 % LK.B ampholines (range 5 to 7), 0-4% ampholines (range 3 5 to 10), 9-5 M-urea and 2-0 % Non-Idet NP-4O, cast in a 1 5 mm x 100 mm tube. The protein samples were dissolved in 9-5 M-urea, 2 % Non-Idet, 5 % 2-mercaptoethanol. Afier prefocusing of the gel, a sample was applied to the basic end and focused for 22 h at 400 V and 1 h at 500 V. For SDSPAGE, an I E F gel was equilibrated with SDS sample buffer, placed across a slab gel (Laemmli, 1970) and electrophoresed as described above. To determine the isoelectric points of relevant polypeptides, a duplicate I E F gel was cut into 5 mm lengths and eluted with o-5 ml deionized water; the p H of the eluted ampholines was measured with a pH meter. These values were plotted with respect to gel length. The isoelectric points were then correlated with the stained bands on another duplicate gel after correction for expansion of the stained/destained gel. Fig. i. Transverse sections of intact and dec (...truncated)