STUDIES ON THE MICROTUBULES IN HELIOZOA V. Factors Controlling the Organization of Microtubules in the Axonemal Pattern in Echinosphaerium (Actinosphaerium) nucleofilum

On the assumption that the double-coiled pattern of microtubules in the axoneme of Echinosphaerium might be due to links of two sizes between adjacent microtubules, we disassembled microtubules with low temperature and then carefully analyzed the patterns of microtubules that formed upon the addition of heat (22 °C) or heat and D20. Although most of the initial clusters of microtubules that formed could not be interpreted as part of an axoneme, the spacings between these microtubules were the same as that in the axoneme, 70 and 300 A. By model building we were able to show that all clusters that form, including stages in the formation of the axoneme and its 12-fold symmetry, could be explained by links of two sizes (70 and 300 A) and the substructure of the microtubule . We could demonstrate these links with improved staining methods . We suggest that nonaxonemal assemblies of microtubules may be eliminated by the natural selection of the most energetically stable configuration of microtubules, all others undergoing disassembly under equilibrium conditions. Model building further supports this suggestion since the model axoneme possesses more links per tubule than any other cluster found . mately these factors must account for the development of the asymmetric form specific for a particular type of cell . In particular, we were interested in investigating the axonemes of Echinosphaerium (Actinosphaerium) nucleofilum, for these structures appear to maintain the asymmetric form of this protozoan . Each axoneme, as demonstrated by Tilney and Porter (1965), is composed of a bundle of microtubules organized, as viewed in transverse section, into two interlocking coils on O cber 0, 2017 jcb.rress.org D ow nladed fom (Figs . 8-11). The tubules are separated from their neighbors in each coil by 70 A and from those in adjacent coils by 300 A . Further, the axoneme itself has 12-fold symmetry, a fact which is particularly obvious in large axonemes . Thus the microtubules, while remaining in the doublecoiled pattern, shaped sectors . We suspected that this highly ordered pattern of microtubules might result, in large measure, from specific types of interactions between adjacent microtubules . Because the separations between adjacent microtubules in the axoneme are almost consistently 70 or 300 A, we were led to inquire if the axoneme in fact may be assembled by interactions between the microtubules brought about by two sizes of macromolecular bridges linking them together . In order to test this supposition we set out to investigate what clues could be obtained by studying the reassembly of microtubules and axonemes after their disassembly with mild agents, such as low temperature (Tilney and Porter, 1967) . It seemed feasible to try to favor the equilibrium conditions for tubule assembly . Under such conditions the microtubule monomer and the postulated linking macromolecules would be induced to assemble rapidly . By careful analysis of the aggregates of tubules thus formed, we hoped that information obtained from "normal" and "abnormal" organizations of microtubules could be interpreted to account for the precise pattern of microtubules in the axoneme. Thus by inducing the organism to rapidly assemble its axonemes we hoped that the earliest aggregates that formed might be uncorrected by whatever mechanisms the cell has at its disposal to eliminate or reorder abnormal patterns, and thus we would be able to obtain information on the factors that normally govern the axonemal pattern . For this purpose we exposed E . nucleofilum to low temperature, 0 °C, for a period sufficient to insure disassembly of all the microtubules (Tilney and Porter, 1967) . We then placed the organisms at room temperature (22 °C) or applied deuterated water (DO) at 22°C. Both D20 and heat are known to favor the polymerized state (Tilney, 1968b) . E. nucleofilum was fixed and processed for electron microscopy shortly after the organism had been placed either at room temperature or in the presence of D20 . Thin sections were then examined for clusters of microtubules . are arranged into 12 wedgeMATERIALS AND METHODS