The Substructure of Ciliary Microtubules

F. D. WARNER 0 1 P. SATIR 0 1 0 University of California , Berkeley, California 1 Department of Physiology-Anatomy , 94720 , U.S.A THE SUBSTRUCTURE OF CILIARY SUMMARY Gill cilia from the freshwater mussel Elliptio complanatus were examined after negative staining with phosphotungstic acid. The doublet and central pair microtubules typically collapse on the grid supporting film so that the protofilaments making up the wall of each tubule spread into a visible array. Thirteen protofilaments can be seen to form both the central pair microtubules and subfibre A of each doublet. Sub fibre B of the doublet consists of 10 protofilaments. As viewed in both normal and thiourea-treated cross-sectioned cilia, subfibre A appears as a complete microtubule with a circular profile and 26 nm diameter. Subfibre B is an incomplete, elliptically profiled tubule that shares, as a common wall or partition, 4 or 5 protofilaments of the A subfibre. The organization of the 35-nm thick protofilaments within the microtubules and their relationship to the various doublet appendages is discussed. INTRODUCTION Inasmuch as microtubules are the main structural component of several important cell organelles, they have elicited interest and controversy concerning both their structure and biochemistry. Early studies of negatively contrasted microtubules from eukaryotic flagella (Andre & Thiery, 1963; Pease, 1963) demonstrated that the wall of individual tubules was made up of a number of linearly arrayed 'protofilaments'. Because of the tendency of microtubules to collapse upon themselves when negatively stained, the number of protofilaments most often observed in single tubules was 4-6. Occasionally as many as 10 filaments were observed at the end of a broken and frayed tubule. The protofilaments were generally observed lying both straight and parallel to one another in both intaci and partially collapsed microtubules, suggesting that the stronger intermolecular bonding was in the linear dimension and that a weaker, colateral bonding held adjacent filaments into the wall lattice. Each protofilament had a diameter of about 4 nm and a repeating 4-nm period occurred along its axis (Grimstone & Klug, 1966). The 4-nm repeat is thought to represent the globular and monomeric form of microtubule protein of molecular weight ~ 60000 (Shelanski& Taylor, 1968). Subsequent observations of sectioned flagella have placed the number of protofilaments per microtubule nearer 12-14 fr both central tubules and doublet subfibre A, and 8-10 filaments for subfibre B (for review, see Arnott & Smith, 1969). Moreover, Phillips (1966) has shown that in the doublet microtubules, subfibre A is a * Present address: Department of Biology, Biological Research Laboratories, Syracuse University, Syracuse, New York, 13210, U.S.A. complete tubule, while subfibre B is an incomplete, C-shaped tubule that shares, according to Ringo (1967), 3 or 4 filaments of the adjacent A subfibre. Recent biochemical studies of doublet microtubules of Chlamydomonas flagella (Witman, 1970; Olmsted, Witman, Carlson & Rosenbaum, 1971) have convincingly shown that each doublet consists of a minimum of 2 proteins or tubulins, both of which are present in each subfibre of the doublet. In addition, certain cytoplasmic microtubules may also consist of 2 tubulins (Bibring & Baxandall, 1971; Bryan & Wilson, 1971; Olmsted et al. 1971). Because of this protein heterogeneity in single microtubules, determination of the precise organization of the tubule wall subunits assumes some significance; particularly in view of the seemingly requisite, periodic attachment sites for the various appendages occurring along the wall of both the A subfibres and the central pair microtubules (Hopkins, 1970; Warner, 1970, 1972; Satir, 1972). In this study we present unequivocal evidence, based on direct observation, that clarifies the number of protofilaments making up the 3 kinds of ciliary microtubules. Furthermore, we present evidence concerning the organization of the protofilaments in the doublet microtubules based on observations of thiourea-treated, 'negatively stained' cilia. MATERIALS AND METHODS Gill cilia of the freshwater mussel Elliptio complanatus were utilized in this study. No distinction is made between the 4 kinds of cilia that occur along the gill epithelium since, with regard to axoneme structure, all are morphologically indistinguishable, although each can be identified by other criteria. For negatively stained preparations, gill cilia were mechanically separated from the epithelium into 10 mM HEPES buffer at pH 7-4. A drop of the cilia-containing suspension was placed on a coated grid and the buffer diluted away with distilled water. A drop of 1 % phosphotungstic acid (PTA) at pH 50 or 7 4 was then added to the grid, excess fluid was removed, and the preparation allowed to air dry. Total staining time was 1-2 min at room temperature. For thin-section microscopy, gill tissue was excised andfixedat 4 C for 1 h in 2 % glutaraldehyde adjusted to pH 7 4 with 005 M sodium cacodylate buffer. The tissue was postfixed for 30 min in 1 % 0sO4 in the same buffer and embedded in Epon 812. Thin sections were stained for 15 min in 5 % aqueous uranyl acetate followed by 2 min in Reynolds' lead citrate. ' Negatively stained' thin-sections (Fig. 10) were prepared by treating the gill tissue with 05 M thiourea for 15 min prior to fixation in 1 % OsO4. Thiourea is generally used as an inhibitor of ciliary motility (Brokaw, 1964). Thin sections were stained as above. All electron micrographs were calibrated to less than 5 % magnification error using carbongrating replicas. OBSERVATIONS AND DISCUSSION Elliptio gill cilia negatively contrasted with PTA are typically observed minus their membrane and with the axoneme splayed apart on the grid supporting film in such a way that the 9 doublet and 2 central microtubules are visible. The doublet tubules generally appear reasonably intact, while subfibre A shows little evidence of its wall subunits (Fig. 2). Subfibre B and the central microtubules are often partially splayed open but, since the tubules tend to collapse upon themselves, no more than 5 or 6 protofilaments are generally visible (Fig. 2). Subfibre A is always distinguishable from subfibre B by its tendency to remain intact or by the presence cf the attached arms and radial links that are sometimes visible. Occasionally, and most often in the distal region of the cilium, the degradative action of the PTA results in the complete collapse of the axoneme microtubules and the spreading of all of their protofilaments into a visible array. Figs. 3-5 show different doublet tubules in this condition and in each, the protofilaments making up the subfibres can be accurately counted. Fig. 4 shows 13 protofilaments in subfibre A, while in the lower region of the micrograph subfibre B is incompletely spread and only 7 filaments are visible. Proceeding distally, however, sub (...truncated)