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ItemOn the mechanism of anaphase spindle elongation in Diatoma vulgare.McDonald, K ; Pickett-Heaps, JD ; McIntosh, JR ; Tippit, DH (Rockefeller University Press, 1977-08)Central spindles from five dividing cells (one metaphase, three anaphase, and one telophase) of Diatoma vulgare were reconstructed from serial sections. Each spindle is made up of two half-spindles that are composed almost entirely of polar microtubules. A small percentage of continuous microtubules and free microtubules were present in every stage except telophase. The half-spindles interdigitate at the midregion of the central spindle, forming a zone of overlap where the microtubules from one pole intermingle with those of the other. At metaphase the overlap zone is fairly extensive, but as elongation proceeds, the spindle poles move apart and the length of the overlap decreases because fewer microtubules are sufficiently long to reach from the pole to the zone of interdigitation. At telophase, only a few tubules are long enough to overlap at the midregion. Concurrent with the decrease in the length of the overlap zone is an increase in the staining density of the intermicrotubule matrix at the same region. These changes in morphology can most easily be explained by assuming zone mechanochemical interaction between microtubules in the overlap zone which results in a sliding apart of the two half-spindles.
ItemAnalysis of the distribution of spindle microtubules in the diatom Fragilaria.Tippit, DH ; Schulz, D ; Pickett-Heaps, JD (Rockefeller University Press, 1978-12)The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.
ItemThe effects of isopropyl N-phenyl carbamate on the green alga Oedogonium cardiacum. I. Cell division.Coss, RA ; Pickett-Heaps, JD (Rockefeller University Press, 1974-10)Cell division in vegetative filaments of the green alga Oedogonium cardiacum is presented as an experimental system. We report on how we have used this system to study the effects of isopropyl N-phenylcarbamate (IPC) on the mitotic apparatus and on the phycoplast, a planar array of cytokinetic microtubules. Polymerization of microtubules was prevented when filaments, synchronized by a light/dark regime and chilled (2 degrees C) while in metaphase or just before phycoplast formation, were exposed to 5.5 x 10(-4) M IPC and then returned to room temperature. Spindles reformed or phycoplasts formed when these filaments were transferred to growth medium free of IPC. However, the orientation of both microtubular systems was disturbed: the mitotic apparatus often contained three poles, frequently forming three daughter nuclei upon karyokinesis; the phycoplast was often stellate rather than planar, and it sometimes was displaced to the side of both daughter nuclei, resulting in a binucleate and an anucleate cell upon cytokinesis. Our results suggest that IPC (a) prevents the assembly of microtubules, (b) increases the number of functional polar bodies, and (c) affects the orientation of microtubules in O. cardiacum. High voltage (1,000 kV) electron microscopy of 0.5-microm thick sections allowed us to visualize the polar structures, which were not discernible in thin sections.
ItemMitosis in the pennate diatom Surirella ovalis.Tippit, DH ; Pickett-Heaps, JD (Rockefeller University Press, 1977-06)Mitosis in Surirella is described; this organism displays a number of unusual features including an unorthodox method of chromosome attachment to the spindle, and the differentiation of an extranuclear central spindle from a large spherical organelle named the microtubule center (MC). The MC, present during interphase, breaks down at late prophase as the central spindle is formed. Later, the spindle enters the nucleus; the chromatin, in association with microtubules (MTs) from the poles, increasingly aggregates around the middle "overlap" region of the central spindle, and by metaphase completely encircles it. Throughout, MTs usually associate laterally with the chromatin. We were not able to identify kinetochore MTs with confidence at either metaphase or anaphase. Instead, at anaphase the leading point of the chromosomes is embedded in a ring of electron-dense material, named the "collar," which encircles each half spindle and extends from the chromatin to the pole. Anaphase separation of the chromosomes is achieved by at least three separate mechanisms: (a) between metaphase and late anaphase the central spindle increases in length by the addition of MT subunits; (b) at late anaphase the central spindle elongates concurrent with a reduction in the overlap; this apparently results from an MT/MT sliding mechanism; (c) each set of chromosomes moves to the poles by a thus far unknown mechanism; however, we anticipate some interaction of the collar and central spindle. At telophase, the polar complexes, (i.e., structures at the spindle pole) separate from the spindle, and later a new MC is formed near each polar complex, after which the polar complexes break down. Aspects of the complex differentiation of the MC, spindle formation, and some unusual characteristics of the diatom spindle as they relate to anaphase motion and spindle function are discussed.