J. Cell Sci. 44, 135-151 (1980) Printed in Great Britain © Company of Biologists Limited 1980

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MICROTUBULES AND CONTROL OF MACRONUCLEAR 'AMITOSIS' IN PARAMECIUM J. B. TUCKER,* J. BEISSON.f D. L. J. ROCHE* 'Department of Zoology, The University, St Andrews, Fife KY16 gTS, Scotland AND J. COHENf f Centre de Ginitique Moliculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France

SUMMARY The ' amitotic' division of the macronucleus during binary fission in P. tetraurelia includes a detailed sequence of shape changes that are temporally coordinated with the adoption of a series of well-defined positions and orientations inside the cell. The deployment of nucleoplasmic microtubules that is spatially correlated with the shaping ritual is more complex and precise than has been reported previously. Macronuclear division is not amitotic. It is not a simple constriction into two halves. As a dividing macronucleus starts to elongate it becomes dorsoventrally flattened against the dorsal cortex of the organism and assumes an elliptical shape. Concurrently, an elliptical marginal band of intranuclear microtubules assembles that has the same spatial relationship to nuclear shape as the marginal microtubule bands of certain elliptical vertebrate blood cells have to cell shape. The band breaks down as further elongation occurs and the nucleus adopts the shape of a straight and slender sausage. Most of the intranuclear microtubules assemble as elongation, starts and break down shortly after elongation is completed; the majority are oriented parallel to the longitudinal axis of the nucleus throughout elongation. Some of them are attached to nucleoli and are coated with granules which are almost certainly derived from the cortices of nucleoli. The peripheral concentration, interconnexion, orientation, and overlapping arrangement of microtubules, and the reduction in microtubule number per nuclear cross-section as elongation proceeds at a rate of about 40 /ttn min"1, are all compatible with the provision of a microtubule sliding mechanism as the main skeletal basis for elongation. There are indications that this mechanism is augmented by anchorage and/or active propulsion of nucleoli that may perhaps facilitate fairly equitable segregation of chromosomal material to daughter nuclei.

INTRODUCTION Division of the highly polyploid varieties of ciliate macronuclei is universally referred to as an amitotic procedure. An amitotic division is one in which a nucleus divides by simple constriction into 2 halves without formation of a spindle or dissolution of the nuclear envelope (Abercrombie, Hickman & Johnson, 1973) and without regular segregation of chromosomal material (Raikov, 1969). The nuclear envelope does remain intact during division of macronuclei, and although these elongating nuclei contain, or are surrounded by, large numbers of microtubules oriented parallel to their longitudinal axes the tubules do not form conventionally constructed mitotic spindles (Carasso & Favard, 1965; Tucker, 1967; Jurand & Selman, 1969, 1970;

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Millechia & Rudzinska, 1971; Stevenson & Lloyd, 1971; Inaba & Kudo, 1972; Walker & Goode, 1976; Jenkins, 1977). However, Raikov (1969) and Grell (1973) have pointed out that it may be incorrect to describe macronuclear divisions as amitotic because of the possibility that genetic information is distributed more precisely during these divisions than was originally supposed. This report supports their views in so far as it establishes that macronuclear division in Paramecium tetraurelia is not just a simple constriction into 2 halves. A detailed and highly coordinated programme of intranuclear microtubule deployment, and nuclear shaping and positioning, is involved. Analysis of macronuclear shaping and positioning is pertinent to the study of 2 widespread but incompletely understood phenomena in cells generally. One is the cytoskeletal basis for precise nuclear positioning and orientation in the cytoplasm of certain cells (for example, Meats & Tucker, 1976). The other, albeit less obvious, is the role of microtubules during control of cell shaping (for review see Tucker, 1979), because this investigation reveals that some aspects of the spatial involvement of microtubules in the shaping of a protozoan nucleus show very close correspondence to those that occur during certain types of metazoan cell shaping. P. tetraurelia is especially favourable material for analysis because of the availability of non-lethal mutants that interfere with these and related phenomena during macronuclear division (Sonneborn, 1974; Beisson & Rossignol, 1975; Ruiz, Adoutte, Rossignol & Beisson, 1976). Such mutants have not been obtained for other cells. This paper establishes a basis for such analysis; it gives a detailed account of the normal course of events during macronuclear division in wild-type P. tetraurelia. It provides evidence for the role of microtubules in shape control that is supported by an accompanying report (Cohen, Beisson & Tucker, 1980) on abnormal microtubule deployment during defective macronuclear division in the tarn 8 mutant of the same organism. MATERIALS AND METHODS

Culture Paramecium tetraurelia stock dd, — 2 (Sonneborn, 1974, 1975) is a derivative of stock 5/ carrying the allele k in the stock J J genetic background. Paramecia were cultured in Scotch Grass infusion which was inoculated with Klebsiella pneumoniae 24 h before inoculation with Paramecium. Electron microscopy Paramecia were prepared for electron microscopy using procedures already described (Tucker, 1967). Organisms at early stages (1-2, see Fig. 1) of binary fission do not have a clearly detectable cleavage furrow and are not readily distinguishable frominterfission organisms when examined using light microscopy after fixation and flat embedding in resin for electron microscopy. Living organisms at stages 1 and 2 possess a slight bulging of the cell body near the mid-region that is detectable when such organisms are examined with a stereo-binocular dissecting microscope. These organisms were isolated individually by pipetting from cultures into fixative and then individually prepared for electron microscopy. Later fission stages were selected on the basis of the stage of development of cleavage furrows (which are well correlated temporally with the progress of nuclear division; see Fig. 1) from cultures fixed during logphase growth that included all stages in the asexual binary fission cycle.

Macronuclear microtubules

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The number and distribution of microtubules in thin cross-sections of dividing macronuclei has been assessed by marking their positions on electron micrographs at final print magnifications of x 30000. Prints were prepared from negatives taken at microscope magnifications of x 15000. Several such negatives and their prints were required to produce a complete cross-sectional montage from negatives at this magnification (which is the minimum, that routinely permitted the sufficiently accurate focusing of the microscope needed to resolve microtubules clearly). Accurate juxtaposition of prints and elimination of 'overlap' during preparation of each montage was achieved by using nucleoli (which can be distinguished from each other on the basis of the different shapes and sizes of their profiles in the sections) as indicators of the margins of regions only included in one negative contributing to a montage. The cross-sections employed were all cut at points along nuclei where their cross-sectional areas were maximal, near the mid-region of each nucleus at stages 2-4, and the mid-regions of putative daughter nuclei (avoiding the narrow central isthmus) at stage 6 (Fig. 1). Light microscopy Additional assessments of changes in the shapes, dimensions, and intracellular positions of macronuclei during the fission cycle, and the temporal correlation of these changes with the other main structural events associated with fission (Fig. 1), are based on light-microscopical examination of fixed organisms prepared using Azure-A, and Dippell's staining procedures (see Cohen et al. 1980). The time elapsing between the start of fission (stage 1) and each succeeding division stage was assessed by examination of individually isolated living paramecia. The progress of changes in cell shape and length was followed for 18 organisms isolated at stage 1 or earlier from log-phase cultures maintained at 27 °C. In some of these organisms changes in the lengths of macronuclei could also be observed.

RESULTS

The main stages in the shaping and positioning of dividing macronuclei are summarized in Fig. 1. The macronuclei of interfission organisms and organisms at 4 stages of division (2, 3, 4 and 6) were examined. The investigation concentrated mainly on stages 3 and 4 (Table 1) because these span the period of most marked elongation and shape change. The shape of an interfission macronucleus approximates to that of a prolate spheroid. No microtubules were detected in interfission nuclei. As fission starts (stages 1 and 2) a macronucleus 'condenses' slightly (Stevenson & Lloyd, 1971). It loses its elongate spheroidal shape (and becomes more compact and spherical) and begins to migrate dorsally and anteriorly from its characteristic interfission position against the gullet. Cross-sections of a stage 2 macronucleus revealed that microtubules had started to assemble in the nucleoplasm and appeared to be randomly oriented. The presence of well-defined anisometric macronuclear organization is first apparent at stage 3. The nucleus is elongate, oriented parallel to the organism's longitudinal axis, and the number of microtubule profiles/nuclear cross-section (N) (Table 1) is much greater (584-825) than it is at stage 2 (190). The percentage of longitudinally oriented (i.e. cross-sectioned) microtubules/nuclear cross-section (as a percentage of JV, see Table 1) lies in the range 72-82%. Serial sectioning revealed

J. B. Tucker, J. Beisson, D. L. J. Roche andj. Cohen

Postfission

Stage 6 18 min

Stage 5 15 min

Fig. i. Nuclear events during the cell cycle of Paramedum tetraurelia. Schematic diagram showing changes in the length, shape and position of a macronucleus (ma) during the cell cycle, including the 6 division stages referred to in the text, based on light- and electron-microscopical examinations. The spatio-temporal relationship of these changes to elongation and cleavage of the cell body, and the positioning of each micronucleus (mi) and oral apparatus (oa) are also shown. The dorsal surfaces of organisms are oriented towards the left side of the diagram and the anterior poles towards the top. The average times elapsing (based on in vivo observations, see Materials and methods) from stage i until each succeding division stage are included. The positions and orientations of the macronuclear cross-sections shown in Figs. 2, 3 and 8 are indicated by short arrows. Stage i. The macronucleus rounds up, micronuclei are at mitotic prophase, a slight bulging of the cortex starts to appear in the anterior portion of the organism and the oral apparatus is already duplicated (Kaneda & Hanson, 1974). Stage 2. The macronucleus migrates towards the anterior portion of the dorsal cortex and the anterior bulging is more marked. The metaphase micronuclei are apparently not confined to any particular cytoplasmic region. Stage 3. The macronucleus becomes dorsoventrally flattened and elliptically shaped (shown in side view here). It elongates from the anterior pole towards the posterior of the organism. The post-telophase micronuclei start to elongate and the new oral apparatus in the opisthe starts to migrate away from the old one. The cleavage furrow is first detectable at this stage. Stage 4. The sausage-shaped macronucleus continues to elongate. Stage 5. The macronucleus starts to constrict in the cleavage furrow plane and pairs of daughter micronuclei transiently take up polar locations. Stage 6. The cleavage constriction is less than 5 /(in across, daughter macronuclei have separated in some organisms and micronuclei lose their apical locations. Postfission.After separation of daughter organisms the macronucleus soon adopts the shape of an elongate spheriod and takes up a position against the oral apparatus again.

Macronuclear microtubules

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Macronuclear truerotubules I

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Fig. 7. Schematic diagram showing the shape and size of the macronucleus at stage 3 with a portion removed to show the cross-sectional profile of the elliptical nucleus at its widest point and the lateral positions of the microtubules of the marginal band with respect to this profile. The elliptical path followed by the band around the periphery of the nucleus is also indicated. The line bearing arrowheads shows the orientation of the organism's longitudinal (polar) axis. The other microtubules in the nucleoplasm at this stage have not been included.

pellicle, and makes contact with the proximal ends of trichocysts attached to the dorsal pellicle (Fig. 2). No cytoplasmic filaments or microtubules that might be involved in producing or maintaining this dorsal positioning by connecting the nucleus to components anchored in the dorsal cortex were detected. Elliptical stage 3 macronuclei contain an elliptically shaped marginal band of microtubules (Figs. 3, 6, 7). Cross-sections of nuclei reveal that each portion of a band includes about 70 microtubules that are more closely packed together than most of the microtubules situated elsewhere in dividing nuclei. Some of them are interconnected by densely staining material (Fig. 6). Between stages 3 and 4 the macronucleus doubles in length (from about 60 /tm up to 120 fim) and adopts the shape of a long and slender sausage (Fig. 1). This phase of elongation is rapid. It was accomplished within 90 s in those living organisms Fig. 3. Cross-section through part of a stage 3 macronucleus grazing through one of its polar-directed extremities so that the marginal microtubule band is sectioned longitudinally, x 58000. Fig. 4. Cross-section of a bundle of peripheral microtubules in a stage 4 macronucleus. Some of the tubules in such bundles are joined together by fine intertubule links (arrow), x 260000. Fig. 5. A granule-coated microtubule cut in cross-section in a stage 3 macronucleus. x 292000. Fig. 6. Cross-section through a portion of a stage 3 macronucleus at one of the lateral extremities of its dor3oventrally flattened cross-sectional profile (compare Fig. 2), showing the compact grouping of the marginal band microtubules. Some of these tubules are joined together by dense material (arrows), x 125000.

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Fig. 8. Cross-section through a stage 4 macronucleus that is closely positioned against the dorsal cortex shown at the same magnification as the stage 3 macronucleus in Fig. 2. The dorsal surface of the nuclear envelope forms several longitudinally oriented pleats (arrows) that are cut in cross-section here, x 7000. Fig. 9. A portion of the dorsally pleated nuclear envelope (