Microtubules and control of macronuclear ‘amitosis’ in Paramecium

J. COHENf 0 1 2 0 f Centre de Ginitique Moliculaire, Centre National de la Recherche Scientifique , 91190 Gif-sur-Yvette , France 1 Fife KY16 gTS , Scotland 2 'Department of Zoology, The University , St Andrews 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. MICROTUBULES AND CONTROL OF 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 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. 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. 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. 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 p (...truncated)