Arrangement of Subunits in Flagellar Microtubules

0 MRC Laboratory of Molecular Biology , Hills Road, Cambridge CB2 2QH, U.K Electron micrographs of outer doublet tubules from flagella have been analysed by methods which make use of the computed diffraction patterns of electron-microscope images. Analysis of singlet A-tubules in the tips of flagella has led to a determination of the helical surface lattice of the A-subfibre, confirming that there are 13 longitudinal protofilaments in the tubule wall and that dimers in neighbouring protofilaments form a staggered arrangement, equivalent to the lattice with an axial periodicity of 8-o nm predicted in earlier work. A low-resolution 3-dimensional image of the A-tubule has been reconstructed, which supports the evidence for an 80-nm-long heterodimer oriented along the protofilaments. The heterodimer is identified as a pair of 4'O-nm morphological units, which appear to be globular at this resolution. 1968; Stephens, 1970). Cytoplasmic microtubules readily dissociate into dimers of ARRANGEMENT OF SUBUNITS IN FLAGELLAR MICROTUBULES A. AMOS AND A. KLUG Filtered images have been obtained from doublet tubules which show that the B-subfibre is also made up of 8 0 - n m dimers, but it differs from the A-tubule in that dimers in adjacent filaments are not in a staggered arrangement but are lined up obliquely at a shallow angle. Using the additional information about the hands of the lattices in the 2 subfibres which is presented in the accompanying paper, a model for the whole doublet has been proposed. The arrangement of the subunits within the outer and central tubules of flagella has been investigated previously by Grimstone & Klug (1966), who analysed electron micrographs of negatively-stained material from Trichonympha and other flagellates using the technique of optical diffraction. They concluded that the subunits comprising the walls of the tubules lie on a helical surface lattice with a period of 4-0 nm along its axis. Additional longitudinal periodicities of 8-o, 16-0 and 48-0 nm which are apparent in the optical diffraction patterns were thought to arise from perturbations in the arrangement of identical subunits and/or by the attachment of other components. A simple displacement in the relative positions of alternate subunits was suggested which would give rise to the observed 8-o-nm periodicity. Since that time, much additional information about the components of cilia and flagella has emerged (for a review see Warner, 1972). In particular, the globular subunits which form the walls of both singlet and doublet tubules are now believed to be molecules of a class of proteins known as tubulins (Mohri, 1968; Stephens, 1970) which are also found in microtubules from other sources (see, for example, Renaud, about 110000 mol. wt. and under more drastic conditions monomers are formed with a molecular weight of approximately 55000. Flagellar tubules dissociate less readily, but under appropriate conditions, their tubulins can be prepared as monomers or dimers. It has been shown that in cytoplasmic tubules and in both A- and B-subfibres of flagellar doublet tubules the tubulin monomers consist of 2 different species in approximately equal amounts (Bryan & Wilson, 1971; Feit, Slusarek & Shelanski, 1971; Olmsted, Whitman, Carlson & Rosenbaum, 1971; Witman, 1970). Because of this Bryan & Wilson have postulated that the dimer of cytoplasmic tubules is a heterodimer of 2 different tubulins. It seems likely that flagellar tubules also consist of heterodimers, since this would give a natural explanation of the strong 8-o-nm period observed in intact tubules from all sources (Pease, 1963; Andre & Thiery, 1963; Grimstone & Klug, 1966; Burton, 1970; Thomas, 1970). Witman, Carlson & Rosenbaum (1972) have found evidence that the 2 main species of tubulin inflagellartubules may be further separated into at least five subspecies, but this result has not yet been confirmed by other workers. The number of longitudinal protofilaments and their arrangement in the doublet tubules has been determined in several species from sectioned material (for example: Ledbetter & Porter, 1964; Phillips, 1966; Ringo, 1967; Warner & Satir, 1973). In most cases it appears that the A-subfibre is a complete tubule consisting of 13 protofilaments, while the B-subfibre is an incomplete C-shaped tubule which has only 10 protofilaments of its own and seems to share the 3 filaments of the A-tubule lying in the midwall of the doublet. The 3 shared filaments form what is known as the partition. These numbers have been confirmed by observations on unfixed, negativelystained material: Erickson (1970)* and Warner & Satir (1973) have independently counted the numbers of protofilaments in favourable micrographs of collapsed and fraying flagellar and ciliary outer fibres. Recent structural studies of embedded and sectioned specimens have clarified the relationship between the tubules and the other structural components of cilia and flagella.' Radial links' or ' spokes' extending from the A-subfibre of each outer doublet into the centre of the axoneme have been found to occur either in pairs about 32-0 nm apart (Warner, 1970; Hopkins, 1970) or in triplets about 24-0 nm apart (Chasey, 19726) along the tubule, with a distance of approximately 96 nm between the centres of the successive groups. Gibbons (1965) demonstrated that the A-subfibres of outer doublets were held together in a cylindrical array by some other component, and Stephens (1970, 1971) and Linck (1973) proposed that there is a component (nexin) bridging A-tubules at intervals of 96-0 nm. The spacing of these 2 components, the radial links and the nexin bridges, appears to determine the longitudinal repeat of the whole flagellum. Optical diffraction patterns of intact axonemes show a 96-o-nm system of layer linesf (R. W. Linck, unpublished work), and we have also found * Paper presented to the 1970 Birmingham meeting of the Society for Experimental Biology. t Layer line spacings referred to in this work, such as 8-o, 16-0 and o.6'O nm, are all based on assigning the value 4-0 nm to the longitudinal spacings of the basic tubulin lattice, which may not be exact for all species. traces of a 96-0-1101 periodicity in computed diffraction patterns of individual tubules. The values reported for the longitudinal spacing of the 2 rows of ' arms' (dynein) on the A-tubule are rather variable, ranging from 12-0 to 24-0 nm (Grimstone & Klug, 1966; Hopkins, 1970; Warner, 1970; Chasey, 19726). However, the clearest pictures (Chasey, 19726; R. W. Linck, unpublished work) show what seem to be single rows of arms with a 24-o-nm spacing. Chasey has suggested that the apparently shorter spacings arise because the 2 rows of arms are not in register. The strong i6-o-nm periodicity seen in the diffraction patterns offlagellartubules is probably due to extra components required to build doublet tubules, possibly accompanied by a perturbation in the packing of the tubules, since i (...truncated)