ABNORMAL MICROTUBULE DEPLOYMENT DURING DEFECTIVE MACRONUCLEAR DIVISION IN A PARAMECIUM MUTANT

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1 J. Cell Sci. 44, (1980) Printed in Great Britain Company of Biologiits Limited ig8o ABNORMAL MICROTUBULE DEPLOYMENT DURING DEFECTIVE MACRONUCLEAR DIVISION IN A PARAMECIUM MUTANT J. COHEN*, J. BEISSON* AND J. B. TUCKERf Centre de Ginitique Moliculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France, and f Department of Zoology, The University, St Andrews, Fife KY16 gts, Scotland 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. INTRODUCTION 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 abnormal (Beisson & Rossignol, 1975; Ruiz, Adoutte, Rossignol & Beisson, 1976). Trichocysts 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 II CEL 44

2 154 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. MATERIALS AND METHODS Strains 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. Culture 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. Light microscopy 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). Electron microscopy The procedures used for electron microscopy were those described by Tucker et al. (1980). RESULTS Macronuclear division 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

3 Defective macronuclear division 155 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 (A c) and tarn 8 organisms (D F) 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 intracellular position varies from one individual to another. It can apparently be located almost anywhere inside the cell. The macronucleus of an interfission wild-type organism is usually somewhat elongate and spheroidal and is located alongside the oral apparatus (Fig. i A). Both tarn 8 and wild-type organisms increase in length during binary fission. There is little time for cell length increase during the rapid (Tucker et al. 1980) elongation of wild-type macronuclei. In terms of its temporal correlation with cell elongation, the elongation of tarn 8 macronuclei takes place more slowly than that of wild-type nuclei, although the time taken for cell division is similar in tarn 8 and wild-type organisms (about 20 min at 27 C). In addition, elongation is less extensive and final lengths are more variable (Fig. 2). In some stage 6 tarn 8 organisms macronuclei had undertaken almost no elongation. The elongation factor (ratio of mean length at late division stages to mean length at early division stages is 3-6 for wild-type organisms and 2-3 for tarn 8 organisms - 64% of the wild-type value). During stage 2 the macronucleus fails to migrate towards the anterior dorsal cortical region. Examination of stained organisms while they are rolled over (see Materials and methods) reveals that during stage 3 macronuclei remain more or less

4 156 J. Cohen, J. Beisson and J. B. Tucker Cell length, Fig. 2. Comparison of macronudear lengths with respect to cell lengths (see Materials and methods) during binary fission in tarn 8 (broken line, open circles) and wild-type organisms (solid line, black circles). The mean values and confidence intervals for cell and macronudear lengths for 5 categories offissionprogress (see below) were calculated. The 'boxes' represent the confidence intervals for each of the means (circles) and the enclosed numbers show the sample sizes for each fission category, p, post fission, immediately after the separation of daughter organisms; 1, interfission; e, early fission (stages 1 and 2); m, middlefission (stages 3 and 4); /, latefission (stages 5 and 6). centrally positioned close to an organism's longitudinal axis and that they do not adopt a flattened elliptical shape against the dorsal cortex as is the case in stage 3 wild-type organisms. Nor does a tarn 8 macronucleus belatedly take up a dorsal position in close contact with the cortex during stage 4 (Fig. 4) or subsequently (Fig. IE). Although dorsoventral flattening does not occur at stage 3, such flattening 200 Fig. 3. Cross-section through a trichocyst tip showing the presence of a sheath of microtubule-like components, x Fig. 4. Cross-section of the dividing macronucleus of a stage 4 organism showing that its cross-sectional area, and the distance separating it from the dorsal cortex (at the top of the figure), are both considerably greater than is the case for wild-type stage 4 organisms (compare with fig. 8 of Tucker et al. (1980) which is also x 7000). Trichocysts (i) are not attached to the dorsal pellicle; many of them are concentrated alongside the macronucleus.

5 Defective macronuclear division 4

6 J. Cohen, J. Beisson andj. B. Tucker Fig. 5. Cross-section of the same macronucleus as that illustrated in Fig. 4 at a point 10 /im. further along its length showing the radical change in the shape of its crosssectional profile, x Fig. 6. Cross-section through a portion of the dividing macronucleus of a stage 4 organism showing several longitudinally oriented folds in the nuclear envelope, x

7 Defective macronuclear division 159 was found along a portion of a stage 4 nucleus (Fig. 5). Microtubules were slightly concentrated at each extremity of the flattened cross-sectional profiles of this portion of the nucleus, but not at the sides of portions exhibiting a more or less circular cross- Fig. 7. Tracings of cross-sections of stage 4 macronuclei in wild-type (A) and tain 8 (B) organisms showing the cross-sectional profiles of their envelopes (dorsal surfaces towards the top of the page) and the positions (black dots) of microtubule crosssectional profiles. sectional profile (Fig. 4). These microtubules were less numerous and less closely packed together than those in the marginal bands of elliptical stage 3 wild-type macronuclei. Cross-sectional areas and the shapes of their cross-sectional profiles vary considerably along the lengths of elongating stage 4 tarn 8 macronuclei (Figs. 4, 5). Such marked variations do not occur in wild-type organisms. Furthermore, very pronounced longitudinally oriented folds in the nuclear envelope were present along some portions of both of the stage 4 nuclei for which sequences of cross-sections were examined (Fig. 6); folding on this scale was never observed for wild-type nuclei.

8 160 J. Cohen, J. Beisson and J. B. Tucker Considerable numbers of microtubules have assembled in the nucleoplasm of elongating nuclei by stage 4. As in wild-type organisms, most of them (79-92%, Table 1) are longitudinally oriented but their distribution is somewhat different. The marked peripheral concentration of microtubules at stage 4 in wild-type macronuclei is not apparent in tam 8 nuclei (Fig. 7). At stage 4 there are 43-67% internal microtubules (microtubules situated more than 1 fim from the nearest portion of the Table 1. Quantitative data based on examination of cross-sections of tam 8 macronuclei Microtubule no. per Cross-sectional Internal Longitudinally cross-section of area of nucleus, microtubules, microtubules, Division stage macronucleus, N fim 1 % % i " S Braces indicate that values were taken from sections of the same organism separated by distances of at least 5 fim along the nuclear longitudinal axis nuclear envelope) (Table 1) compared with 15-48% in wild-type organisms. As for wild-type macronuclei, some of the peripheral microtubules are coated with dense granules. Whether tubules are also attached to nucleoli was not ascertained; extensive sequences of longitudinal thin sections of elongating tarn 8 macronuclei were not prepared. Correlated with the slower, less extensive, and more variable elongation of tarn 8 macronuclei compared with those of wild-type organisms are the greater and more variable cross-sectional areas of tarn 8 macronuclei, and the substantially greater numbers of microtubule profiles per nuclear cross-section (iv), during the period (stages 4-6) of most marked macronuclear elongation (Table 1, Figs. 7, 8). For example, at stage 4 the cross-sectional areas of tarn 8 nuclei lie in the range /tm 2 with a range of for N, while in wild-type organisms these values are fim 2 and , respectively. The value of N decreases as macronuclei elongate in both tarn 8 and wild-type organisms. Decreases during stage 5 and 6 (after most of the elongation of wild-type nuclei is completed) may be largely a consequence of microtubule breakdown, since macronuclei contain few microtubules as fission is completed (Fig. 8). One of the stage 4 tarn 8 macronuclei had a much higher number of microtubules per cross-section than the maximum value recorded for wild-type nuclei (Fig. 8). It is not clear whether this is an indication that a greater than normal number of microtubules assemble in tarn 8 macronuclei or if iv rises briefly to such high values in wild-type organisms at a stage (between 2 and 3) that has not been examined ultrastructurally.

9 Defective macronuclear division b Division stages Fig. 8. Changes in macronuclear cross-sectional area (squares) and the number of microtubules per cross-section (circles) during macronuclear division in tam 8 (broken lines) and wild-type organisms (solid lines) based on data included in Table i of this paper and table i of Tucker et al. (1980). Each point on the graphs represents a value taken from a single organism; they are mean values in cases where a macronucleus was examined at 2 regions along its length (see Tables). Points showing values for area and micro tubule number from the same organism are situated vertically above and below one another. For clarity, in cases where more than one organism at a particular division stage has been examined, the values recorded have been spread to the right with respect to the abscissal calibration of division stages in order of decreasing microtubule number, on the assumption (see text) that such decrease represents progress from one stage to the next. During stages 5 and 6 the tam 8 macronucleus usually fails to narrow and then divide at its mid-point; it is pinched in 2 by the advancing cleavage furrow. The constricted portion forms a 'nuclear bridge' (Fig. IF). Occasionally, however, a macronucleus may undergo a more or less central thinning out, similar to that which occurs in wild-type organisms (Fig. ic) before the cleavage furrow is sufficiently

10 162 J. Cohen, J. Beisson and J. B. Tucker advanced to exert a pinching action. Furthermore, the macronucleus is often not positioned medially with respect to the poles of an organism, so that the cleavage furrow plane does not coincide with the mid-point of the nucleus. As a result segregation of macronuclear material to daughter organisms can be very unequal (Fig. 9). In extreme cases of such 'slippage' one of the daughter organisms does not receive a portion of a macronucleus and an 'amac' cell results. Considerable numbers of amac cells are always present in log-phase cultures of tarn S10 6 I Hi TflnD_ % macronuclear length in proter % macronuclear length in proter Fig. 9. Comparison of the proportion of macronuclear material in proters (putative anterior daughters), represented as a % of total macronuclear length situated anterior to the cleavage furrow (see Materials and methods), during stages 5 and 6 in wild-type (A) and tarn 8 organism (B). In both cases a sample of 37 organisms was examined and the percentage values were grouped into 5 % range classes (starting 0-5 %). Other microtubular systems Since the tarn 8 lesion interferes with microtubule organization in the defectively dividing macronucleus, does it also perturb microtubule organization and activity in the cell's other microtubular systems? Ciliary beating and ultrastructure appear to be normal. Although trichocysts do not exhibit saltatory movement (Aufderheide, 1978) and their tips do not attach to the plasma membrane in tarn 8, microtubules seem to be arranged (Fig. 3) in a manner identical with that described for trichocyst tips in P. caudatum (Bannister, 1972). Each micronucleus elongates extensively during binary fission in a wild-type organism as it produces a long (up to 80 ju,m) intranuclear microtubular separation spindle (Jurand & Selman, 1970; Stevenson & Lloyd, 1971). This separation spindle is very unusual; it consists mainly of tubules with diameters of up to 32 nm in thin-sectioned material (Tucker, 1979). Microtubules in the macronucleus and cytoplasm have diameters of about 24 nm. This peculiarity is also manifested by the separation spindles of tarn 8 micronuclei (Fig. 10) which, unlike tarn 8 macronuclei, do not

11 :&m Defective macronuclear division Fig. 10. Cross-section through the 2 micronuclear separation spindles of a stage 4 organism. Most of the spindle tubules have diameters in the range nm. Compare with the 24-nm-diameter cytoplasmic microtubule (arrow), x r A TOOr o 0 T No. of micronuclei/cell or more No. of micronuclei/cell Fig. 11. Comparison of the number of micronuclei/cell in wild-type (A, 66 cells) and tarn 8 organisms (B, 93 cells) based on examination of organisms stained with Azure A. All organisms were of the same clonal age; 14 fissions following autogamy. All organisms possess 2 micronuclei immediately following autogamy (Sonneborn, 1974). differ in any marked way from those of wild-type organisms (in terms of crosssectional area or microtubule number, spacing, and arrangement). Nor did examination of organisms stained with Azure A reveal any delay or reduction in the elongation of micronuclear separation spindles. However, segregation of daughter micronuclei is substantially impaired in tarn 8 organisms; 56% of the interfission organisms examined contained more, or less, than the normal pair of micronuclei (Fig. 11).

12 164 J. Cohen, J. Beisson andj. B. Tucker DISCUSSION Microtubules and macronuclear division The dividing macronuclei of tarn 8 organisms elongate less extensively and more slowly than those of wild-type organisms. Correlated with this, the intranuclear microtubules are not peripherally concentrated in tarn 8, and the numbers of microtubules per nuclear cross-section persisting at relatively late division stages (4 and 5) are about the same as those at an earlier stage (3) in wild-type organisms prior to the main rapid phase of elongation. These findings are compatible with other evidence (Tucker et al. 1980) that elongation is promoted by a peripherally situated microtubulesliding mechanism. Reduced elongation in tarn 8 may also be partly due to delay in elongation. Some tarn 8 nuclei stop elongating although putative daughter nuclei have not separated. Disassembly of microtubules at stage 6 may intervene to reduce still further the potential of the microtubules for involvement in nuclear elongation. The failure of tarn 8 macronuclei to adopt an elliptical shape during stage 3 is another indication that intranuclear microtubules are abnormally deployed in these nuclei, since such shaping is spatially correlated with a marginal microtubule band in wild-type organisms (Tucker et al. 1980). The tarn 8 lesion and microtubules In tarn 8 organisms trichocyst motility and positioning are abnormal, micronuclear segregation is irregular, and the shaping and positioning of macronuclei are affected especially during binary fission. Why does the tarn 8 lesion produce this range of defects? Examination of dividing macronuclei indicates that nucleation of microtubule assembly inside nuclei and subsequent elongation of these tubules are not impaired to any marked degree. The apparently normal organization of microtubules in cilia, around trichocyst tips, and in micronuclear separation spindles also suggests that the mutation does not have a direct effect on the assembly or basic structure of the organism's microtubules. Possibly the genetic lesion interferes with the action of microtubule-associated contractile components. Intracellular motility in general involves a variety of ATPases such as dynein and actomyosin complexes, as well as a range of proteins that regulate the force-generating activities of these molecules (Goldman, Pollard & Rosenbaum, 1976; Dustin, 1978; Stebbings & Hyams, 1979; Roberts & Hyams, 1979). Different forms of motility within a single cell may exploit a different selection of these components. It may be that the action of only one such component is directly affected by the tarn 8 lesion, so that micronuclear elongation and ciliary action proceed normally while macronuclear elongation and shaping, and the saltation and transport of trichocysts to the cell surface are impaired. The irregular segregation of micronuclei may be due to malfunction of a procedure that is not directly associated with microtubules (see below).

13 Defective macronuclear division 165 The tarn 8 lesion and cortical control of nuclear division There is an alternative way of accounting for the defects inflicted by the tarn 8 mutation. The lesion may interfere with cell-surface-mediated interactions. In tarn 8 organisms the dividing macronucleus does not take up a dorsal subcortical position yet the longitudinal orientation of elongating macronuclei and their abnormally distributed nucleoplasmic microtubules still occurs. Hence, such orientation is not dependent on subcortical positioning, although it is possible that adoption of an elliptical shape (which does not take place in tarn 8) and formation of an elliptical marginal band of microtubules requires the close nucleo-cortical association that is achieved only in wild-type organisms. Interfission tarn 8 macronuclei also have abnormal shapes and positions. The positioning of other organelles is abnormal. Trichocysts do not exhibit saltatory motion (Aufderheide, 1978) and cortical attachment. In wild-type organisms pairs of daughter micronuclei become localized at opposite poles after breakdown of separation spindles and before the completion of cleavage (Tucker et al. 1980). This localization may ensure the even segregation of daughter micronuclei in wild-type organisms that is lacking in tarn 8. Since micronuclear elongation proceeds normally in tarn 8 it is perhaps subcortical localization that is defective. Hence the mutation is perturbing events at the cortex, in the general cytoplasm, and within the macronucleus. Is the primary defect in some cytoplasmic or cortical component? Are all the nuclear abnormalities a consequence of defective nucleo-cortical interactions rather than a direct intranuclear effect of the tarn 8 lesion? This is a distinct possibility bearing in mind the pronounced nucleo-cortical interactions reported for other ciliates. Macronuclear shaping, division, and DNA synthesis are sensitive to the state of the cortex in Stentor (De Terra, 1978). Analysis of Tetrahymena mutants reveals that cortical development and macronuclear division are morphogenetically related, as are construction of the cortical oral organelles and micronuclear division (Frankel, Jenkins & De Bault, 1976). Furthermore, micronuclear spindles make close contact with the cortex in dividing Tetrahymena (Jaeckel- Williams, 1978). If the tarn 8 mutation exerts its effect only via macronucleo-cortical interactions then these influence the spatial arrangement of the intranuclear microtubules. What sort of signals might be involved and how could they cross the nuclear envelope? A variety of cytoskeletal networks extend from the vicinity of nuclei to the cell surface in a range of cell types during interfission (see Weber, Pollack & Bibring, 1975). It has been suggested that intermediate filaments are involved in the control of nuclear positioning; they still form an extensive network during cell division (Blose, 1979). There is experimental evidence for cytoskeletal interactions between nuclei and plasma membranes (Wang, Gunther & Edelman, 1975; Berke & Fishelson, 1979). A mutation that affects cytoskeletal components connecting trichocysts and nuclei to plasma membranes might disturb control of macronuclear division, trichocyst/plasma membrane attachment, and polar localization of micronuclei. No such connexions between macronucleus and cortex have been detected in wild-type organisms (Tucker et al.

14 166 J. Cohen, J. Beisson andj. B. Tucker 1980) but they may be of a type that is not preserved during preparation for electron microscopy, such as discrete regions of subsurface cytoplasmic gelation. It is especially interesting that the same positional defects (for both macro- and micro-nuclei) as those described above for tarn. 8 are caused by a number of other mutations at different chromosomal loci which all prevent attachment of trichocysts to the plasma membrane (Cohen & Beisson, submitted for publication). This is further evidence for positional interactions between the cell surface, nuclei and trichocysts in Paramecium. We thank Mr D. L. J. Roche for skilful assistance with electron microscopy. A training fellowship from the Ligue Nationale Francais contre le Cancer to J. C, support from the Delegation Generale a la Recherche Scientifique et Technique (grant no ) to J.B., and from the Science Research Council (U.K.) to J.B.T. are gratefully acknowledged. REFERENCES AUFDERHEIDE, K. J. (1978). Genetic aspects of intracellular motility: cortical localization and insertion of trichocysts in Paramecium tetraurelia. J. Cell Sci. 31, BANNISTER, L. H. (1972). The structure of trichocysts in Paramecium caudulum. J. Cell Sci. 11, BEISSON, J. & ROSSIGNOL, M. (1975). Movements and positioning of organelles in Paramecium aurelia. In Nucleocytoplasmic Relationships during Cell Morphogenesis in some Unicellular Organisms (ed. S. Puiseux-Dao), pp Amsterdam: Elsevier. BERKE, G. & FISHELSON, Z. (1976). Possible role of nucleus-membrane interaction in capping of surface membrane receptors. Proc. natn. Acad. Sci. U.S.A. 73, BLOSE, S. H. (1979). Ten-nanometer filaments and mitosis: maintenance of structural continuity in dividing endothelial cells. Proc. natn. Acad. Sci. U.S.A. 76, COHEN, J. & BEISSON, J. Genetic analysis of the relations between the cell surface and the nuclei in Paramecium tetraurelia. (Submitted to Genetics, Princeton.) DALAMATER, E. D. (1951). A staining and dehydrating procedure for the handling of microorganisms. Stain Technol. 26, DE TERRA, N. (1977). Some regulatory interactions between cell structures at the supramolecular level. Biol. Rev. 53, DIPPELL, R. V. (1955). A temporary stain for Paramecium and other protozoa. Stain Technol. 30, DUSTIN, P. (1978). Microtubules. Berlin, Heidlberg and New York: Springer. FRANKEL, J., JENKINS, L. M. & DE BAULT, L. E. (1976). Causal relations among cell cycle processes in Tetrahymenapyriformis. An analysis employing temperature-sensitive mutants. J. Cell Biol. 71, GOLDMAN, R., POLLARD, T. & ROSENBAUM, J. (1976). Cell Motility. Cold Spring Harbor Conferences on Cell Proliferation. Vol. 3. GRANT, P. (1978). Biology of Developing Systems. New York and London: Holt, Rinehart and Winston. JAECKEL-WILLIAMS, R. (1978). Nuclear divisions with reduced numbers of microtubules in Tetrahymena. J. Cell Sci. 34, JURAND, A. & SELMAN, G. G. (1970). Ultrastructure of the nuclei and intranuclear microtubules of Paramecium aurelia. J. gen. Microbiol. 6o, ROBERTS, K. & HYAMS, J. S. (1979). Microtubules. New York and London: Academic Press. Ruiz, F., ADOUTTE, A., ROSSIGNOL, M. & BEISSON, J. (1976). Genetic analysis of morphogenetic processes in Paramecium. I. A mutation affecting trichocyst formation and nuclear division. Genet. Res. 27, SONNEBORN, T. M. (1970). Methods in Paramecium research. In Methods in Cell Physiology, vol. 4 (ed. D. M. Prescott), pp New York: Academic Press.

15 Defective maaronuclear division 167 SONNEBORN, T. M. (1974). Paramecittm aurelia. In Handbook of Genetics, vol. 2 (ed. R. C. King), pp New York: Plenum. Press. STEBBINGS, H. & HVAMS, J. S. (1979). Cell Motility. London and New York: Longman. STEVENSON, I. & LLOYD, F. P. (1971). Infrastructure of nuclear division in Paramecium aurelia. I. Mitosis in the micronucleus. Aust. J. biol. Sri. 24, TUCKER, J. B. (1979). Spatial organization of microtubules. In Microtubules (ed. K. R. Roberts & J. S. Hyams), pp New York and London: Academic Press. TUCKER, J. B., BEISSON, J., ROCHE, D. L. J. & COHEN, J. (1980). Microtubules and control of macronuclear 'amitosis' in Paramerium. jf. Cell Sci. 44, WANG, J. L., GUNTHER, G. R. & EDELMAN, G. M. (1975). Inhibition by colchicine of the mitogenic stimulation of lymphocytes prior to S phase. J. Cell Biol. 66, WEBER, K., POLLACK, R. & BIBRINC, T. (1975). Antibody against tubulin: the specific visualization of cytoplasmic microtubules in tissue culture cells. Proc. natn. Acad. Sri. U.S.A {Received 5 February 1980)

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