Abnormal microtubule deployment during defective macronuclear division in a Paramecium mutant

0 Centre de Ginitique Moliculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France, and f Department of Zoology, The University , St Andrews, Fife KY16 gTS, Scotland J. COHEN*, J. BEISSON* AND J. B. TUCKERf SUMMARY The tarn 8 mutant of Paramecium tetraurelia is a representative of a class of mutants characterized by abnormal nuclear divisions during binary fission and the failure of trichocysts to attach to the plasma membrane. Compared with wild-type organisms the following abnormalities occur in tarn 8 individuals. (1) The spherical interphase macronucleus is not positioned near the oral apparatus; it is randomly located in the cytoplasm of interfission organisms. (2) The macronucleus does not migrate towards the anterior dorsal cortex as its division starts, nor is it dorsally and subcortically positioned as it elongates. (3) Elongating macronuclei exhibit variable and irregular shapes. (4) This elongation is delayed and reduced. (5) Longitudinally oriented microrubules assemble in the nucleoplasm of dividing macronuclei but their spatial deployment is abnormal. (6) Unequal segregation of micronuclei between daughter organisms occurs during binary fission. The abnormal arrangement of nucleoplasmic microtubules provides support for the proposal that a microtubule sliding mechanism is involved during the elongation of dividing macronuclei. The extent to which macronuclear division may be controlled by the cell cortex is considered in relation to the pleiotropic efFects of the tarn 8 mutation. - Cell division requires precise spatial and temporal adjustment between nuclear and cytoplasmic division. Asymmetric divisions yielding cell products of unequal size, such as occur during budding in yeast or during 'oblique' spindle construction and associated spiral cleavage in various invertebrate blastomeres (Grant, 1978), provide striking evidence for specific control of the positioning of dividing nuclei within cells. However, the mechanisms by which such control is effected remain obscure. This problem can be approached with Paramecium by studying mutants in which cytoplasmic division proceeds normally although nuclei are aberrantly positioned and an unequal partitioning of nuclear material between daughter cells results. Most of these Paramecium mutants belong to a particular class that consistently display 2 main phenotypic abnormalities: both nuclear and trichocyst positioning are abnorcysts are secretory vesicles that normally attach to certain plasma membrane ' docking sites' where they remain until discharge is stimulated. In these mutants the trichocysts never attach to the plasma membrane and the dividing macronucleus never J- Cohen, J. Beisson and J. B. Tucker reaches the normal dorsal subcortical position described in the accompanying paper (Tucker, Beisson, Roche & Cohen, 1980). This paper analyses defective macronuclear division during binary fission in the mutant tarn 8 (a representative of the class of mutants discussed above) in terms of spatio-temporal correlations between abnormal positioning, defective elongation, abnormal and irregular shaping, and abnormal nucleoplasmic microtubule deployment, for the dividing macronucleus. In addition, the arrangement of microtubules in the tips of abnormally positioned trichocysts and the separation spindles of dividing micronuclei (that become unequally distributed between daughter organisms) are described. These examinations appear to rule out the possibility that the tarn 8 mutation exerts its influence by interfering with microtubule assembly in a marked fashion. It is argued that the pleiotropic effects of the tarn 8 mutation indicate that considerable spatial control of macronuclear division is exercised by the cell cortex. The mutant tarn 8 (Beisson & Rossignol, 1975) was isolated after nitrosoguanidine treatment of stock 1I4-2 of P. tetraurelia and first screened on the basis of its lack of trichocyst discharge. The trichocysts are normal in shape but fail to attach to the plasma membrane and do not exhibit the saltatory motion (Aufderheide, 1978) that is performed by trichocysts prior to plasma membrane attachment in wild-type organisms. Paramecia were cultured using procedures described by Sonneborn (1970) at 27 C in Scotch Grass infusion or cerophyl infusion supplemented with /?-sitosterol (0-4 fig/mi). Infusions were inoculated with Klebsiella pneumomae 24 h before inoculation with Paramecium. Organisms were isolated from log-phase cultures, transferred to microscope slides, fixed by adding one drop of Dippell's (1955) stain and then examined using bright-field microscopy. These preparations were not covered with coverslips, so that organisms could be gently rolled over in the drops by blowing a jet of air at them to ascertain the location of the macronucleus (central or subcortical). Cell lengths, macronuclear lengths, and the lengths of the portions of macronuclei situated in proters (in cases where a cleavage furrow was visible) were measured using an ocular micrometer. Micronuclei and micronuclear spindles were examined using phase-contrast microscopy in organisms stained with Azure A (Dalamater, 1951). The procedures used for electron microscopy were those described by Tucker et al. (1980). A detailed comparison of macronuclear division in tarn 8 with that which occurs in stock d<f-2 of the wild-type organism (see Tucker et al. 1980) from which this mutant is derived has been undertaken. The division stages referred to below have been distinguished, using the criteria described by Tucker et al. (1980), on the basis of changes in the lengths and shapes of organisms and the progress of cleavage furrowing. These cortex-associated changes all appear to proceed normally in tarn 8. The fine structure of 4 tarn 8 organisms has been examined (2 at stage 4 and 1 each at stages 5 and 6) to ascertain the spatial organization of microtubules in the macronucleus and monitor changes in its cross-sectional area during the period of most marked nuclear elongation. Fig. 1. Phase-contrast micrographs (x 250) of Paramecia prepared using Dippell's stain showing the shapes and positions of macronuclei in wild type (Ac) and tarn 8 organisms (DF) at several stages in the fission cycle. The anterior poles of organisms are oriented towards the top of the figure. A. Wild type, interfission. B. Wild type, division stage 2. C. Wild type, stage 5, the organism's dorsal cortex is to the left of the figure. D. tarn 8, interfission organism with spherical macronucleus. E. tarn 8, stage 6, the elongating macronucleus is not dorsally positioned, F. tarn 8, stage 6 shortly before the separation of daughter organisms. The macronucleus is not positioned medially in the organism, with respect to its poles. Fission of the 'macronuclear bridge' between daughters is effected by the cleavage furrow. In an interfission tarn 8 organism the macronucleus is more or less spherical (Fig. i D) and its intracel (...truncated)