Ciliary inter-microtubule bridges

F. D. Warner 0 0 Department of Biology, Biological Research Laboratories, Syracuse University , Syracuse, New York 13210 , U.S.A SUMMARY Electron micrographs of both negatively contrasted and thin-sectioned lamellibranch gill cilia reveal several new features of ciliary fine structure, particularly in regard to those structures forming intermittent or permanent crossbridges between microtubules. Negativecontrasting reveals the presence of a 145-nm repeating bridge between the central microtubules. Frontal views of negatively contrasted dynein arm rows along subfibre A show that the arms (23-nm repeat) in the outer row are displaced in a left-handed manner by 3-4 nm with respect to those in the inner row. This displacement is probably a direct reflexion of the helical tubulin subunit lattice of the subfibre. Interdoublet (nexin) links are seen connecting adjacent A and B subfibres at intervals of 86 nm along the doublet. Negative-contrasting shows thin, highly elastic connexions holding the doublets together. When seen in longitudinal thin sections, the interdoublet links are often tilted to considerable angles, indicating they may have an elastic response to interdoublet sliding. - The motile 9 + 2 axoneme of cilia, flagella and sperm tails is an interconnected lattice of microtubules having no fewer than 5 kinds of structures forming either intermittent or permanent crossbridges between microtubules (see Warner, 1974, for 1973) have clearly shown that the generative force for flagellar motility results from active (ATP hydrolysis) crossbridge formation between adjacent doublet microtubules. mechanochemical interaction results in sliding displacement between the doublets which is simultaneously converted into propagated bending motion. Recently, we examined the role of the radial spokes in the sliding-bending conversion (Warner & Satir, 1974) and concluded that the spokes, in lamellibranch gill cilia, were the primary source of the internal shear resistance required to convert active interdoublet sliding into regulated, propagated bending of the organelle. Although this study presented many new details of ciliary fine structure, the observations were primarily limited to the radial spoke-central sheath complex. Using both thin-sectioned and negatively contrasted, isolated gill cilia, it is now possible to visualize the presence of a bridge between the 2 central microtubules, to clarify the organization of the interdoublet or nexin links and to characterize the organization of the 2 rows of dynein arms along the doublet subfibre A. MATERIALS AND METHODS Lamellibranch gill cilia from the genus Unio were used in this study. For electron microscopy, gill tissue was isolated and fixed in 2 % glutaraldehyde adjusted to pH 7-4 with 25 mM sodium cacodylate for 1-5 h at 4 C. Tissue was postfixed in cacodylate-buffered 1 % OsO4 for 45 min, dehydrated in an ethanol series and embedded in Epon 812. Thin sections were stained for 15 min in 5 % aqueous uranyl acetate followed by staining for 2 min in Reynolds' lead citrate. For some preparations, gill tissue was deciliated and the cilia purified by differential centrifugation. Purified cilia were demembranated in Triton X-100 and either fixed as above or negatively contrasted with 2 % aqueous uranyl acetate at pH4'5. Isolated axonemes are functionally intact; that is, they will reactivate and beat normally upon the addition of ATP. Details of the isolation, purification and reactivation procedure will be published in a future paper. OBSERVATIONS AND DISCUSSION The movement of axonemal microtubules during ciliary or flagellar beating must be regarded as a very dynamic process, since each doublet microtubule slides with respect to every other doublet and with respect to the central microtubule complex, even though the major part of this sliding may be only a passive response to the active sliding generated between any 2 doublets at a particular instant during the beat cycle. Since we know that each linear element moves with respect to all other linear elements (Warner, 1972; Warner & Satir, 1974), all structural crossbridges between microtubules, whether intermittent or permanent, must be brought into the conceptual framework of the sliding filament model of ciliary motility (Satir, 1968). Lamellibranch gill cilia have a prominent 'bridge' which occurs between doublet numbers 5 and 6 and forms the main morphological marker for determining orientation of the cilium (Gibbons, 1961; Satir, 1965, 1968). The bridge appears to be a manifestation of arm structure between these doublets, that is, the arms of both rows of doublet 5 appear to be permanently crossbridged to doublet 6. The bridge is readily apparent in transversely sectioned cilia (Figs, i, 11-14) and individual elements repeat at 23 nm along the A subfibre (Warner & Satir, 1974). Our previous study of lamellibranch gill cilia (Warner & Satir, 1974) described in detail the organization of the central sheath-microtubule complex. The sheath consists of paired rows of projections along each of the 2 central microtubules to which are attached, intermittently, the radial spoke heads. Longitudinal thin sections suggested the presence of a periodic bridge between the central tubules (as have numerous other studies, e.g. Gibbons, 1961) but superimposition of the sheath projection rows could also account for the bridged appearance, particularly since the measured periodicity is the same for both projections and the bridge region. When seen in transverse sections, the central sheath projections, although not clearly resolved, form a circular profile around the central tubules (Fig. 1) and there also appears to be material or a bridge joining the 2 tubules at the axoneme axis. This bridge spans the 8-nm space between tubules and often appears to be double. However, a straightforward answer regarding the presence of a central microtubule bridge is provided by negatively contrasted images of the central complex. Fig. 2 shows an intact central tubule-sheath complex and again it is not possible to separate the image of the sheath projections from the bridge region. Fig. 3 shows a central pair with most of the sheath material solubilized except that along 1 tubule, a single doublet remains attached to the sheath via the triplet radial spokes. Fig. 4 shows a central pair with all sheath material removed: clearly there remains a prominent 14'5-nm repeating bridge between the 2 tubules. Fig. 5 shows a similar preparation except that the central pair has pulled apart and the bridge elements remain attached to only one of the tubules. Although it appears from these preparations that only a single row of bridge elements occurs between the central tubules, the 9ame arguments that apply to possible dynein arm superimposition (see next section) also apply here. All motile 9 + 2 cilia, flagella and spermtails have an ATPase, dynein, located in the 2 orderly rows of arms of subfibre A of ea (...truncated)