A Mutation Affecting Basal Body Duplication and Cell Shape in Paramecium

The thermosensitive mutant sm19 of Paramecium tetraurelia undergoes a progressive reduction in cell length and basal body number over successive divisions at the nonpermissive temperature of 35°C. In spite of these defects, sin19 cells retain the same generation time as wild-type cells at 35°C. Cytological observations at both electron and light microscopy levels reveal no other perturbation than the rarefaction of basal bodies and the rare (3%) absence of one or two microtubules in basal bodies or ciliary axonemes. The temperature-sensitive period, during the last 30 min of the cell cycle, corresponds to the phase of basal body duplication. Upon transfer back to the permissive temperature, all basal bodies are normally duplicated. The mutational defect is transiently restored by microinjection of wild-type cytoplasm or of a soluble proteic fraction from wild-type cell homogenates. Altogether, the cytological and physiological data support the conclusion that the sm/9 + gene codes for a diffusible product required for the initiation of basal body duplication and would thus be the first identified gene involved in this process. Our data also indicate that in Paramecium basal body number is not coupled with control of the cell cycle, but helps determine the shape of the cell via the organization of the cytoskeleton. T HE basal bodies of ciliates and flagellates duplicate in the same way as centrioles: each new basal body develops close to a preexisting one and at right angles to it. The factors that trigger duplication and the mechanisms that control the assembly of this ubiquitous eukaryotic structure are still unknown, but could, in principle, be genetically dissected provided that mutations blocking some step of the process could be phenotypically identified. Ciliates provide a favorable system for detection of defective basal body duplication, since their cortex is made of highly ordered cortical rows of regularly spaced basal bodies, easily observed under light microscopy. Mutants with reduced numbers of basal bodies have indeed already been described in Paramecium (26) and Euplotes (14). However, in none of these cases has it been established that the mutation primarily affected basal body duplication per se, rather than subsequent events involving the insertion of basal bodies in the plasma membrane. We describe here a thermosensitive nuclear recessive mutation of Paramecium tetraurelia, sml9, which results, over successive divisions at the nonpermissive temperature, in a progressively decreasing number of basal bodies and a correlatively reduced cell length and modified cell shape. By following different cytological and physiological parameters (cell length, basal body number, cortical organization, ultrastructure, generation time, protein synthesis, and feeding activity), we have analyzed the expression of the mutation upon transfer of sm19 cells to the nonpermissive temperature and their recovery after return to the permissive temperature. All the observations support the conclusion that the primary effect of the mutation affects basal body duplication. We have also demonstrated that the mutational defect can be repaired transiently by microinjection of soluble cytoplasmic factors ofa proteinic nature. The results will be discussed in relation to the mechanisms of basal body duplication and to the role of basal body number in the control of the cell cycle and the determination of cell shape. Materials and Methods Strains and Culture Conditions The wild-type strain used in these experiments and from which the mutant sml9 was isolated was the stock d4-2 of P. tetraurelia (39). Depending on the experiments, the wild-type strain and the mutant expressed either one of the twa complementary mating types, referred to as VII and VIII (38, 40). We also used the mutants sin2 and sm3 isolated from stock 51 of P. tetraurelia by Jones and Berger (26) and kindly provided by Dr. J. Berger, University of British Columbia, Vancouver, British Columbia, Canada. These two thermosensitive mutants are characterized by an abnormal cell shape and a reduced number of basal bodies at the nonpermissive temperature, 35°C. The mutation nd7(3, 40), which prevents trichocyst discharge, was used as a genetic marker in certain crosses. Cells were grown at either 28 or 35°C in a grass infusion bacterized with Aerobacter aerogenes and supplemented with 0.4 gg/ml 13-sitosterol. Mutagenesis and Crosses Mutagenesis was carded out with N-methyl,N'-nitro-N-nitrosoguanidine from Aldrich Chemical Co. at a final concentration of 75 ~tg/ml for 45 min © The Rockefeller University Press, 0021-9525/87/03/417/14 $1.00 The Journal of Cell Biology, Volume 104, March 1987 417--430 417 on O cber 8, 2017 jcb.rress.org D ow nladed fom on exponentially growing cells competent for autogamy. After mutagenesis, autogamy was induced by starvation. This leads to the breakdown of the old macronucleus and to the formation of new microand macronuclei homozygous for all their genes, so that ex-autogamous cells can express the mutations that have been induced. In this experiment, 14,000 cells were isolated in 96-well plates as previously described (8) and all the clones were tested at 35°C by replica plating after 10 generations at 28°C. All the clones presenting morphological abnormalities were kept for further investigation. Crosses were carried out according to the classical methods described by Sonneborn (39). Synchronization of the Cells From exponentially growing populations maintained at 280C for 10 generations after a controlled autogamy, pools of 25 cells in the last stage of division were picked up and transferred into fresh medium at either 28°C or at the restrictive temperature (35°C) using slides and medium preincubated at the respective temperatures. Within each pool, all the ceils completed their division within a 5-min interval, yielding a small population of synchronous cells at the time 0 of their next cell cycle. Members of a synchronous pool were either isolated or kept together in depression slides and periodically examined for observation of the successive divisions. At chosen times, cells were picked up and fixed for length measurements or cytological observations. Measurements of the Cells Measurement of cell length was chosen as the simplest and most significant parameter for the mutants studied, Cells were gathered in a drop of culture fluid and fixed by addition of a drop of Dippelrs stain (11), a rapid technique previously shown to preserve the in vivo dimensions (Beisson, J., and M. Rossignol, unpublished observation). Length was measured with an ocular micrometer adapted on a Zeiss light microscope at low magnification without a coverslip. Depending on the experiment, cells were measured just after completion of division or 1 h later. Cytological Techniques Silver Impregnation. The method was essentially that of Chatton and Lwoff (7). lmmunocytoiogical Methods. Immunodecoration of specific cortical structures was carried out using the Schliwa and van Blerkom method (37) adapted to Paramecium as described by Cohen et al. (9). Deciliation was obtained immediately before permeabilization treatment by two successive transfers into 10 mM MnC12 (15) until immobilization. The antitubulin antiserum used was that raised against Paramecium axonemal tubulin described by Cohen et al. (9). After permeabilization, cells were incubated for 1 h in the antiserum diluted 1/400, washed twice for 5 min, then incubated for 1 h in FITC-antirabbit antibodies diluted 1/200. Ceils were rinsed twice, mounted in glycerol containing 2 % n-propyl-gallate to reduce photobleaching of the fluorochromes (16), observed, and photographed under a Zeiss epifluorescent microscope. Electron Microscopy. The cells were fixed in 2% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) for 90 min at 4°C. After washing in the same buffer, the samples were postfixed in 1% osmium tetroxide in 0.05 M cacodylate for 60 min. The samples were then dehydrated by passage through a series of ethanol and propylene oxide baths and embedded in Epon. Thin sections were contrasted with ethanolic uranyl acetate and lead citrate, then examined with a Philips EM 201 electron microscope. Scanning Electron Microscopy. The cells were quickly deciliated in MnCI2 and fixed in 2% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 30 min. In this medium, the cells were then attached to polylysinetreated coverslips (33) by centrifugation for 10 rain at 3,000 rpm. After washing in 0.05 M cacodylate buffer, the attached cells were postfixed in 1% osmium tetroxide for 30 min in the same buffer and further dehydrated in a graded series of ethanol and acetone. The samples were dried at the critical point and examined with a Phillips 505 scanning electron micro-