Half Spindles Research Articles

A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression.

A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.

Meiosis, spindle pole body cycle, and taxonomy of the heterobasidiomycetePachnocybe ferruginea

Meiosis and the meiotic spindle pole body cycle were studied electron microscopically in basidia of the heterobasidiomycetePachnocybe ferruginea. Spindle pole body splitting in prometaphase I and II, and intermeiotic and postmeiotic duplication were investigated in particular detail. During prophase, the spindle pole body consists of two three-layered discs connected by a middle piece. At late prophase I and again in prometaphase II, the discs contact the nuclear envelope. Then, the nuclear membrane at the contact area is separated from the non-contacted part of the nuclear envelope and finally disappears. Each disc nests into the nuclear opening of the otherwise intact nuclear envelope. The disc remains in the gap and generates a half spindle. At late metaphase I, a co-disc develops eccentrically within the parent disc. The co-disc detaches from the parent disc during interphase I and becomes one of the metaphase II spindle pole bodies. Co-discs are absent during the second division. A cap of endoplasmic reticulum encloses each disc during prophase I through anaphase I. In the second meiotic division, the caps covering the spindle pole bodies of one nucleus of the pair, are developed from the neighbouring nucleus. Spindle pole bodies ofP. ferruginea are similar to those of the rusts, and especially to those ofEocronartium muscicola andHelicobasidium mompa.

Development of the quadripolar meiotic cytoskeleton in spore mother cells of the moss Funaria hygrometrica

ABSTRACT Evidence presented in the accompanying paper that plastids function as microtubule (MT)-organizing centres for development of the quadripolar cytoskeleton of pre-meiotic spore mother cells (SMCs) in the moss Funaria hygrometrica is complemented here by observations on the MT system in these cells. Early in meiotic prophase numerous MTs align progressively along the two plastids as they elongate. Concomitant with (and perhaps causal for) plastid rotation, new MT arrays grow from each tip of each plastid to both tips of the other plastid. The ‘along-plastid’ and ‘between-plastid’ arrays ultimately form the edges of a tetrahedron, enclosing the prophase nucleus. MT breakdown at the centre of each edge leaves four cones of MTs, one emanating from each vertex, located at the plastid tips. These partially fuse in between-plastid pairs to give a twisted spindle with broad knifeedge poles oriented at right angles to one another, i.e. a condensed form of the quadripolar precursor. The twist causes the metaphase plate and the subsequent phragmoplast and organelle band to be saddle-shaped, and the daughter nuclei to be elongated perpendicular to one another along the two knife edges. The tetrahedral array returns during interkinesis and again breaks down into four cones of MTs centred on the plastid tips; these, however, now become individual half spindles for the two perpendicularly arranged second division spindles. When meiosis is completed the four haploid nuclei thus come to lie at the vertices of a tetrahedron that was established by MT-mediated plastid positioning during meiotic prophase. The tetrahedral cage of MTs precedes meiosis yet predicts the planes of division, and in these two respects it is the meiotic counterpart of the preprophase band of MTs, which develops before mitosis in most higher plant cells.

Localization of sea urchin egg cytoplasmic dynein in mitotic apparatus studied by using a monoclonal antibody against sea urchin sperm flagellar 21S dynein

AbstractA monoclonal antibody against sea urchin (Hemicentrotus pulcherrimus) sperm flagellar 21S dynein was characterized and sued to identify and localized cytoplasmic dynein of sea urchin eggs by the methods of immunoblotting and indirect immunofluorescence microscopy. D57, the monoclonal antibody used in this study, was directed to the Aβ polypeptide of 21S dynein. D57 stained sperm flagella specifically but did not inhibit Mg‐ATPase activity of 21S dynein, its recombination ability with NaCl‐extracted axonemes, or the movement of demembranated sperm. D57 cross‐reacted with sea urchin egg cytoplasmic dynein. High molecular weight cytoplasmic dynein polypeptide which had the same electrophoretic mobility s flagellar dynein. A chains was the only polypeptide that reacted with D57 in the crude extract from unfertilized sea urchin eggs. Indirect immunofluorescence observations showed that the mitotic apparatus was stained most intensely in the frozen sections and lysed eggs. In the mitotic apparatus isolated at metaphase, the half spindles were stained more strongly than the astral regions. The regions near chromosomes in the half spindle appeared to be stained particularly. Staining of the interzone was also observed in the mitotic spindle isolated at anaphase. Comparison of the staining patterns for cytoplasmic dynein with that for tubulin suggested that cytoplasmic dynein was localized where microtubules were densely organized, but its distribution may not necessarily be identical with that of microtubules.

Reorganization of microtubules during mitosis in Dictyostelium: Dissociation from MTOC and selective assembly/disassembly in situ

AbstractWe applied the “agar‐overaly” immunofluorescence techinque (Yumura, S., H. Mori, and Y. Fukui, J. Cell Biol. 99:894–899, 1984) to a semisynchronous culture of Dictyostelium discoideum for studying the organization changes in the microtubule system during mitosis. Using a flurescent DNA dye DAPI (4′,6′ ‐diamidino‐2‐phenylindole), chromatin fibers and individual chromosomes were visible in cells prepared by this method, whereby the mitotic phase could be critically evaluated.We found that a rapid shortening of the cytoplasmic microtubules was preceded by a structural dislocation from their organizing centers (MTOCs) in the midprophase, resulting in the transient occurrence of free microtubules in the cytoplasm. Statistic analyses showed that microtubule disassembly in prophase was diphasic. Initially long, wavy microtubules shortened from their distal ends. Following dissociation of their proximal ends from the MTOC, all microtubules initiated rapid disassembly, probably from both ends. During this process, microtubule assembly from the now duplicated spindle pole body (SPB) resumed.This study also revealed novel information on the dynamics of the Dictyostelium mitotic spindle: 1) Half spindles interdigitate in the spindle center, and the extent of interdigition increases coincidentally with the spindle elongation, and 2) during the anaphase to telophase, a subpopulation of spindle microtubules elongates while the rest of the microtubules disasemble very rapidly.Overall this study indicates the presence of elaborate mechanisms responsible for the selective assembly/disassembly of particular microtubule subpopulations in situ.

Spindle function in the green alga Mougeotia: Absence of anaphase A correlates with postmitotic nuclear migration

AbstractIn the filamentous green alga Mougeotia, each daughter nucleus formed by mitosis is then rapidly moved along the recently divided daughter cell to the central cleavage developing in the chloroplast. This movement is brought about by a cone‐shaped array of microtubules (MTs) that ensheath the daughter nucleus and are focused upon a small region, presumably a microtubule‐organising center (MTOC). Movement is completed when the MTOC locates and then resides in the chloroplast cleavage, drawing the nucleus into this position.The mitotic spindle is open with initially broad, ill‐defined poles. Anaphase A contributes minimally, if at all, to chromosome separation since the half spindles remain about the same length during anaphase and telophase. Thus, anaphase is accommodated and probably achieved by spindle elongation; the interzonal MTs also generate a rudimentary phragmoplast at the ingrowing cleavage furrow. The persistent polar MTs become directly transformed into the cone‐shaped array and initiate nuclear movement during early telophase. Various closely or distantly related green algae show this trait of persistent polar MTs. We conclude that this trait has allowed some species to evolve a motility system based directly on the capabilities of astral MTs, for generating the postmitotic nuclear movement essential for the restoration of the interphase cell organization.

Metabolic inhibitors and mitosis: III. Effects of dinitrophenol on spindle disassembly inPinnularia

At telophase in the diatomPinnularia, the two half spindles that comprise the central spindle, separate and then disassemble unidirectionally from the end formerly in the central overlap, back to the pole (Soranno andPickett-Heaps 1982). The metabolic inhibitors dinitrophenol plus deoxyglucose were applied to cells at telophase, depleting their ATP levels at the early stages of half-spindle disassembly; the cells were maintained in this state for 5 minutes, before the inhibitors were washed out. Disassembly of the half spindles, as judged from their birefringence, ceased in ATP-depleted conditions, and recommenced soon after the inhibitors were removed, going to completion quite rapidly.

Characterstics of motor innervation of muscle spindles in the monkey

The motor nerve supplies of four whole muscle spindles and 16 half spindles (equator and one pole) from lumbrical muscles of the monkey were reconstructed by light microscopy of serial, 1-microm-thick transverse sections.The 24 poles of spindle were innervated by 37 fusimotor (gamma) axons and 18 skeletofusimotor (beta) axons. Sixty-seven percent of spindle poles received gamma axons only, 25% were supplied by both gamma and beta axons or beta axons only, and 8%were not innervated by motor axons. All beta axons except one and 35% of the gamma axons innervated one type of intrafusal fiber only. The other 65% of gamma axons coinnervated two or three different types of intrafusal fiber. Gamma axons innervated the (dynamic) bag1, intrafusal fiber together with the (static) bag2,and or chain fibers in 65% of the poles of spindle in which the bag1 fiber received motor supply. The bag2 fiber shared gamma-innervation with either the bag1 or chain fibers in nearly every spindle pole. The chain fibers were usually coinnervated with the bag2 fiber by gamma axons. Compared to spindIes in the cat tenuissimus muscle, the monkey spindle received fewer gamma axons and had a higher incidence of shared gamma-innervation between the dynamic and static intrafusal fibers. Unlike cat tenuissimus spindles, the monkey spindles lacked gamma axons selective to the bag2 fiber. However, the beta motor system within the monkey lumbrical muscle was organized in a manner similar to the cat tenuissimus muscle. The significance of these observations is discussed relative to the general motor organization and function of spindles in different muscles and species of mammals.

A calcium-rich intraspindle membrane system in spermatocytes of wolf spiders.

The meiotic spindle of spermatocytes of two wolf spiders contains a highly organized system of ER-like membranes. In cells observed ultrastructurally at early prometaphase, these membranes completely invest each bivalent and are present in the periphery of the spindle in association with the centrosomes. By metaphase each bivalent and its kinetochore fibers are completely encased in a tube of this membrane. We have treated living spermatocytes with the permeant, fluorescent-chelate probe, chlorotetracycline (CTC) to determine whether or not the intraspindle membrane system is rich in associated Ca2+. Spider testes were dissected into PIPES-buffered saline containing 200 microM CTC and were kept in this solution for 10 min. Autofluorescence controls were prepared by incubation in saline without CTC, and nonspecific effects of CTC were assessed by incubation for 10 min in 200 microM oxytetracycline (OTC). Neither unstained nor OTC-treated spermatocytes emit significant fluorescence. In contrast, CTC treatment yields bright, punctate fluorescence, which coincides with the distribution of the mitochondria. The plasma membrane is only weakly fluorescent, while the nuclear envelope exhibits prominent fluorescence. The chromosomes are not fluorescent during prophase, but after nuclear envelope breakdown, they become outlined by dim, but distinct fluorescence. As spindle formation commences, the CTC signal from the intraspindle membrane system becomes strong. In some cells, thin lines of CTC fluorescence are apparent in the metaphase half spindle; this fluorescence pattern mimics the distribution of the intraspindle membrane system and suggests that it is rich in associated Ca2+. We suggest that the intraspindle membrane system functions in the regulation of cytosolic Ca2+ during meiosis through sequestration of the cation.

Control mechanisms of the cell cycle: role of the spatial arrangement of spindle components in the timing of mitotic events

To characterize the control mechanisms for mitosis, we studied the relationship between the spatial organization of microtubules in the mitotic spindle and the timing of mitotic events. Spindles of altered geometry were produced in sea urchin eggs by two methods: (a) early prometaphase spindles were cut into half spindles by micromanipulation or (b) mercaptoethanol was used to indirectly induce the formation of spindles with only one pole. Cells with monopolar spindles produced by either method required an average of 3 X longer than control cells to traverse mitosis. By the time the control cells started their next mitosis, the experimental cells were usually just finishing the original mitosis. In all cases, only the time from nuclear envelope breakdown to the start of telophase was prolonged. Once the cells entered telophase, events leading to the next mitosis proceeded with normal timing. Once prolonged, the cell cycle never resynchronized with the controls. Several types of control experiments showed that were not an artifact of the experimental techniques. These results show that the spatial arrangement of spindle components plays an important role in the mechanisms that control the timing of mitotic events and the timing of the cell cycle as a whole.

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum I. Prophase I — Prometaphase I

A thoroughly documented account of the ultrastructure of the meiotic spindle pole body (SPB) cycle in a rust (Basidiomycota, Uredinales) is presented for the first time. The three-dimensional structure of the SPB and spindle during meiosis in the hollyhock rust fungusPuccinia malvacearum is analyzed from serial sections of preselected stages. This paper covers prophase I to prometaphase I. At late prophase I, the nucleolus disperses and does not reappear until the end of meiosis. The SPB at late prophase I consists of two, 4-layered discs, 0.8–1.0 μm in diameter, connected by a middle piece (MP). The SPB is associated with a differentiated region of the nuclear envelope and nucleoplasm. At late diplotene to diakinesis, each disc generates a half spindle as it inserts into an otherwise intact nuclear envelope. The MP connecting the interdigitating half spindles elongates and eventually splits transversely during subsequent spindle elongation. Each half MP, which is attached to a SPB disc, becomes inserted in a sheath-like extension of the nuclear envelope. The intranuclear late prometaphase I spindle always becomes oriented perpendicularly to the longitudinal axis and sagittal plane of the metabasidium. There are 200–290 spindle microtubules (MTs) at each SPB at late prometaphase. The nonkinetochore MTs form a coherent central spindle around which the kinetochore MTs and bivalents are spread. A metaphase plate is absent. The results are compared with SPB behavior and spindle structure in early meiosis of other basidiomycetes and ascomycetes.

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum

Changes in the spindle pole body (SPB) and meiotic nuclei from interphase I through interphase II in the hollyhock rustPuccinia malvacearum are analyzed ultrastructurally by three-dimensional reconstructions from serial sections. Interphase I nuclei undergo a coordinated migration and rotation during which the SPBs approach the convex face of the lateral promycelial wall. During the transition from interphase I to prometaphase II, the collateral disc (co-disc) apparently enlarges and fuses with the main disc of the SPB. The resulting single SPB nucleates two confluent half spindles and about 225 astral microtubules (MTs). Co-discs and middle pieces (MPs) are absent during division II. SPBs separate and form metaphase II intranuclear spindles oriented in a predictable manner. Tubular cisternae are present within the spindle at early metaphase II. The architecture of the spindle at division II is essentially identical to that reported for division I except that the spindle is about half as long. Anaphase-telophase II nuclear envelope constriction, separation of the sibling nuclei, and externalization of the SPBs is identical to that reported for division I. Genesis of the duplicated interphase II SPB apparently occurs rapidly and involves formation of the MP followed by the three-layered SPB discs. General aspects of the division II spindle are discussed. A model for the meiotic SPB cycle in a rust is presented and its phylogenetic and functional significance in relation to other basidiomycetes and ascomycetes is discussed.

Nonrandom chromosome segregation in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural study.

During meiosis I in males of the mole cricket Neocurtilla (Gryllotalpa) hexadactyla, the univalent X1 chromosome and the heteromorphic X2Y chromosome pair segregate nonrandomly; the X1 and X2 chromosomes move to the same pole in anaphase. By means of ultrastructural analysis of serial sections of cells in several stages of meiosis I, metaphase of meiosis II, and mitosis, we found that the kinetochore region of two of the three nonrandomly segregating chromosomes differ from autosomal kinetochores only during meiosis I. The distinction is most pronounced at metaphase I when massive aggregates of electron-dense substance mark the kinetochores of X1 and Y chromosomes. The lateral position of the kinetochores of X1 and Y chromosomes and the association of these chromosomes with microtubules running toward both poles are also characteristic of meiosis I and further distinguish X1 and Y from the autosomes. Nonrandomly segregating chromosomes are typically positioned within the spindle so that the kinetochoric sides of the X2Y pair and the X1 chromosome are both turned toward the same interpolar spindle axis. This spatial relationship may be a result of a linkage of X1 and Y chromosomes lying in opposite half spindles via a small bundle of microtubules that runs between their unusual kinetochores. Thus, nonrandom segregation in Neocurtilla hexadactyla involves a unique modification at the kinetochores of particular chromosomes, which presumably affects the manner in which these chromosomes are integrated within the spindle.

Microtubule turnover as a mechanism of mitosis and its possible evolution

Movement of chromosomes during mitosis appears to be coupled to the unidirectional turnover of spindle microtubules. This paper outlines a directional turnover model of mitosis which hypothesizes that: (1) Unidirectional turnover of tubulin subunits and microtubule-associated proteins occurs from an assembly site at one end to a disassembly site at the other end of mitotic apparatus microtubules. (2) The components of interpolar microtubules are continuously moving toward each pole due to their assembly in the opposite half spindle and disassembly in the near half spindle. (3) Mitotic chromosome movements are coupled to this directional turnover by selective formation of semi-stable lateral interactions or bridges between kinetochore microtubules and parallel interpolar microtubules. (4) The anaphase velocity of kinetochores relative to the poles is determined by the rate that kinetochore microtubules disassemble on their poleward ends minus the rate they assemble at their kinetochore ends. (5) Spindle elongation occurs when assembly of interpolar microtubules is more rapid than their disassembly or when interpolar microtubules slide in an anti-parallel arrangement. (6) The velocity of chromosome separation is the sum of spindle elongation and the poleward movements of sister chromosomes. Evidence for and against these hypotheses and some possible steps in the evolution of this type of mechanism are discussed.

Behavior of kinetochores during mitosis in the fungus Saprolegnia ferax.

In rapidly growing hyphae of Saprolegnia ferax, all nuclei contain arrays of kinetochore microtubules, which suggests that the nuclei are all in various phases of mitosis, with no apparent interphase. In prophase nuclei, kinetochore microtubules form a single, hemispherical array adjacent to the centrioles. This array separates into two similar arrays after centriole replication. The two arrays form by separation of the initial group of microtubules, with no kinetochore replication. During metaphase, between 6.5 and 85% of the kinetochores occur as amphitelic pairs, with a slight tendency for pairing to increase as the spindle elongates. 100% pairing has never been observed. The interkinetochore distance in these pairs is consistently similar to or approximately 0.17 microns. Throughout metaphase and early anaphase, there is extensive and increasing diversity in kinetochore microtubule length, so that a true metaphase plate has not been found. During metaphase, anaphase, and telophase, kinetochore numbers vary considerably, with a mean of similar to or approximately 30 per half spindle. A number of artefactual causes for this variability were examined and discarded. Thus, these results are accepted as real, suggesting either variable ploidy levels in the coenocytic hyphae or kinetochore replication during mitosis.

Nuclear cycle of Saprolegnia ferax.

The mitotic nuclear (equivalent to cell) cycle of the oomycete fungus, Saprolegnia ferax, was analysed by quantitative serial-section electron microscopy of hyphal nuclear populations synchronized by inhibition of DNA synthesis by fluorodeoxyuridine (FdUrd). Following telophase and karyokinesis, kinetochore mitrotubules persist into G1 stage as a single group of approximately 42 per nucleus (2n = 42 for this species). During G1 the centrioles replicate and kinetochore microtubules separate into 2 groups of approximately 21, a configuration they retain through S and G2. During metaphase a new population of kinetochore microtubules are formed, each one of an amphitelic pair connecting to the opposite pole to that associated with the persistent microtubule from the previous division. Thus, by the end of metaphase, there are approximately 42 kinetochore microtubules per half spindle. FdUrd, applied for 2 h with uracil, completely blocks DNA synthesis yet permits centriole replication and causes nuclei to accumulate with 2 pairs of centrioles, 2 arrays (each of 21) of kinetochore microtubules, and apparently enlarged nucleoli. Removal of FdUrd permits rapid (within 30 min) DNA synthesis followed by successive rounds of decreasingly synchronous nuclear cycles. These post-FdUrd cycles are 2.5 times longer than normal at 2.5 h, with S plus G2 being more extended than other phases. Calculated durations of a normal nuclear cycle are: G1, 33 min; S, 7 min; G2, 10 min; metaphase, 8 min; anaphase, 0.5 min; and telophase, 4 min.

Mitosis in the pennate diatom Surirella ovalis.

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.

Birefringence and Ultrastructure of Isolated, Stabilized Spindles

Previous investigations on the spindle apparatus in primary spermatocytes of the crane fly, Nephrotoma suturalis, with polarizing optics have shown that chromosomal fibers can be detected as positively birefringent bands extending from the chromosomes to the poles (1). Chromosomal fiber birefringence reaches a maximum at metaphase when five distinct fibers can be seen in each half spindle. An obvious question raised by these results was what is the ultrastructural basis of birefringence?Initial attempts to characterize the ultrastructure of crane-fly spindles showed that there were hundreds of microtubules (MTs) in these spindles, but there was no evidence that they were distributed in a pattern that corresponded to the pattern of birefringence (2,3).

THE GLYCEROL-ISOLATED MITOTIC APPARATUS: A RESPONSE TO PORCINE BRAIN TUBULIN AND INDUCTION OF CHROMOSOME MOTION.

The birefringence of the MAs or spindles isolated from sea urchin eggs with the 1 M glycerol-isolation medium was stabilized when more than 0.5 mg/ml tubulin was contained in the medium. The addition of glycerol up to a final concentration of of 4 M strongly stabilized the MAs even in the absence of GTP and tubulin. The birefringence of the spindle and asters was not reduced even for the periods of several hours. The incorporation of heterogeneous tubulin into the isolated anaphase MAs was demonstrated by augmentation of the birefringence at the interzonal region as well as half spindles accompanied by enlargement of spindle and asters. In the anaphase MAs isolated in the absence of brain tubulin, chromosomes moved a short distance toward the poles upon addition of ATP, Mg2+ and 0.5 mg/ml tubulin. When the MAs were isolated in the presence of 0.5 mg/ml tubulin, the chromosomes moved in a more regular fashion to half the way to the poles accompanied by an increase in spindle length by 10 to 15%. GTP could not be substituted for ATP for inducing the motion. The chromosome motion of the isolated anaphase spindle was less significant than that of the isolated MA. Increasing tubulin concentration to 3 mg/ml, the chromosomes in the isolated MA separated at random by an unusual growth of the spindle. The stretch of the interzonal region by incorporating heterogeneous tubulin seemed to push the chromosomes apart abnormally. It was suggested that brain tubulin in a range of 0.5 mg/ml supports a tubulin-MA microtubule equilibrium favoring more regular motion of chromosomes in vitro.

Cell division in the pennate diatom Diatoma vulgare.

Mitosis and cytokinesis in the pennateDiatoma is described. Prior to division, a doubled “Persistent Polar Complex” (PPC), the focus of numerous cytoplasmic microtubules, migrates from near the nucleus to one side of the cell near the girdle bands, followed by the nucleus. The central dense core of the doubled PPC breaks down as a central spindle grows between the two PPCs, which now each become characteristically associated with a small vacuole and other features of unknown significance. The tubules of the central spindle terminate in a layer, the “Spindle Insertion” (SI), close to the PPCs; other, “polar” tubules radiate from each SI, mostly toward the nucleus, which becomes increasingly deformed by them until the nuclear envelope is ruptured. The elongating spindle enters one side of the nucleus laterally; as shown previously, the central spindle consists of two interdigitated half spindles, but the polar tubules which diverge laterally from the SI, by metaphase form a complex, cone-shaped array emanating from each pole. Some polar tubules penetrate the chromatin and may represent true kinetochore tubules; others, which persist conspicuously throughout mitosis, extend tangentially past the chromatin, out into the cytoplasm, intersecting with those from the other pole. The core structure of the PPCs alters from being plate-shaped at prophase, to being rod-like by metaphase. The chromosomes form a donut-shaped mass of chromatin penetrated by the central spindle throughout meta-and anaphase. Chromosomal separation is accomplished in two stages: the chromatin splits and moves up to the SI, and then the central spindle elongates, concurrent with its overlap region decreasing markedly in extent. Thus, this latter part of anaphase movement could be generated by microtubule sliding past microtubule. Then each PPC distinctly separates from its SI, and moves away from the daughter nucleus during cytokinesis; it again becomes the focus of numerous tubules and often ends up in one corner of the daughter cell. Meanwhile, the central spindle and the SIs, now surmounted by the reforming telophase nuclei, slowly disintegrate during cleavage.