Half Spindles Research Articles

From fertilization to cancer: the role of centrosomes in the union and separation of genomic material.

Centrosomes play crucial roles in the union of sperm and egg nuclei during fertilization and in the equal separation of genomic material during cell division. While many studies in recent years have focused on the molecular composition of centrosomes, this article focuses on the structural behavior of centrosomes and on factors that play a role in centrosome functions under normal, artificially altered, and abnormal conditions. We review here how studies in the classic sea urchin egg model have contributed to our knowledge on the centrosome cycle within the cell cycle, on compaction and decompaction of centrosomal material, and on the contributions of maternal and paternal centrosomes during fertilization. Centrosome material is activated in unfertilized eggs by increasing pH with ammonium and by increasing calcium with the ionophore A23187, which are conditions that are normally induced by sperm. D(2)O and taxol also induce centrosome aggregation in the unfertilized egg. Maternal and paternal centrosome material both contribute to the formation of a functional centrosome but the formation of a bipolar centrosome requires material from the paternal centrosome. Fertilization of taxol-treated eggs reveals that the male centrosome possesses the capability to attract maternal centrosome material. When pronuclear fusion of the male and female pronuclei is inhibited with agents such as the disulfide reducing agent dithiothreitol (DTT) a bipolar mitotic apparatus is formed from the paternal centrosome. Furthermore, one centrosome of the bipolar mitotic apparatus is capable of organizing an additional half spindle that attaches to the female pronucleus indicating a functional and perhaps structural connection between centrosomes and chromatin. Sea urchin eggs are also useful to study centrosome abnormalities and consequences for the cell cycle. While classic studies by Theodor Boveri have shown that dispermic fertilization will result in abnormal cell division because of multiple centrosomes contributed by sperm, abnormal cell division can also be induced by chemical alterations of centrosomes. Compaction and decompaction of centrosome structure is studied using chloral hydrate or the chaotropic agent formamide, which reveals that centrosomes can be chemically altered to produce mono- or multipolar abnormal mitosis and unequal distribution of genomic material upon release from formamide. The patterns of abnormal centrosome reformations after recovery from formamide treatment resemble those seen in cancer cells which argues that structural defects of centrosomes can account for the formation of abnormal mitosis and multipolar cells frequently observed in cancer. In summary, the sea urchin model has been most useful to gain information on the role of centrosomes during fertilization and cell division as well as on adverse conditions that play a role in centrosome dysfunctions and in disease.

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence.

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against alpha-tubulin, gamma-tubulin, and 4'-6'-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two "first polar bodies" and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster.

XMAP230 is required for normal spindle assembly in vivo and in vitro.

XMAP230 is a high molecular mass microtubule-associated protein isolated from Xenopus oocytes and eggs, and has been recently shown to be a homolog of mammalian MAP4. Confocal immunofluorescence microscopy revealed that XMAP230 is associated with microtubules throughout the cell cycle of early Xenopus embryos. During interphase XMAP230 is associated with the radial arrays of microtubules and midbodies remaining from the previous division. During mitosis, XMAP230 is associated with both astral microtubules and microtubules of the central spindle. Microinjection of affinity-purified anti-XMAP230 antibody into blastomeres severely disrupted the assembly of mitotic spindles during the rapid cleavage cycles of early development. Both monopolar half spindles and bipolar spindles were assembled from XMAP230-depleted extracts in vitro. However, spindles assembled in XMAP230-depleted extracts exhibited a reduction in spindle width, reduced microtubule density, chromosome loss, and reduced acetylation of spindle MTs. Similar defects were observed in the spindles assembled in XMAP230-depleted extracts that had been cycled through interphase. Depletion of XMAP230 had no effect on the pole-to-pole length of spindles, and depletion of XMAP230 from both interphase and M-phase extracts had no effect on the rate of microtubule elongation. From these results, we conclude that XMAP230 plays an important role in normal spindle assembly, primarily by acting to stabilize spindle microtubules, and that the observed defects in spindle assembly may result from enhanced microtubule dynamics in XMAP230-depleted extracts.

The cytoplast concept in dividing plant cells: cytoplasmic domains and the evolution of spatially organized cell division

The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single "tensegral" unit. The term "tensegrity" is a contraction of "tensional integrity" and the concept proposes that the whole cell is organized by an integrated cytoskeleton of tension elements (e.g., actin fibers) extended over compression-resistant elements (e.g., MTs).During cell division, a primary role of the spindle is seen as generating two cytoplasts from one with separation of chromosomes a later, derived function. The telophase spindle separates the newly forming cytoplasts and the overlap between half spindles (the shared edge of two new domains) dictates the position at which cytokinesis occurs. Wall MTs of higher plant cells, like the MT cytoskeleton in animal and protistan cells, spatially define the interphase cytoplast. Redeployment of actin and MTs into the preprophase band (PPB) is the overt signal that the boundary between two nascent cytoplasts has been delineated. The "actin-depleted zone" that marks the site of the PPB throughout mitosis may be a more persistent manifestation of this delineation of two domains of cortical actin. The growth of the phragmoplast is controlled by these domains, not just by the spindle. These domains play a major role in controlling the path of phragmoplast expansion. Primitive land plants show different morphological changes that reveal that the plane of division, with or without the PPB, has been determined well in advance of mitosis.The green alga Spirogyra suggests how the phragmoplast system might have evolved: cytokinesis starts with cleavage and then actin-related determinants stimulate and positionally control cell-plate formation in a phragmoplast arising from interzonal MTs from the spindle. Actin in the PPB of higher plants may be assembling into a potential furrow, imprinting a cleavage site whose persistent determinants (perhaps actin) align the outgrowing edge of the phragmoplast, as in Spirogyra. Cytochalasin spatially disrupts polarized mitosis and positioning of the phragmoplast. Thus, the tensegral interaction of actin with MTs (at the spindle pole and in the phragmoplast) is critical to morphogenesis, just as they seem to be during division of animal cells. In advanced green plants, intercalary expansion driven by turgor is controlled by MTs, which in conjunction with actin, may act as stress detectors, thereby affecting the plane of division (a response clearly evident after wounding of tissue). The PPB might be one manifestation of this strain detection apparatus.

The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a "sliding filament mechanism" during the formation and function of the mitotic spindle.

In vitro maturation of transported pig oocytes in a defined protein-free medium

The duration of the different phases of the microtubule and chromosome cycles were estimated in the native diploid cell populations of Allium cepa L root meristems proliferating undisturbed, under steady state conditions, at the physiological temperature of 15°C. The cycles were coupled by considering their fitting in relation to the short process of nuclear envelope breakdown. In the cycle related to cytoplasmic division, the preprophase band which predicts the future position of the phragmoplast made its appearance, as a wide band, 16 mm before the G2 to prophase transition, ie it was only present during the final 5% of the total G2 timing (5 h 30 mm). The band became narrow only 6 mm after prophase had started and it was present in this form for the remaining prophase time (2 h 24 mm). Its disappearance occurred strictly coinciding with nuclear envelope breakdown, at the end of prophase. No microtubules related to cytoplasmic division were apparent until 9 mm after telophase had initiated. The two initial stages of phragmoplast formation which followed occupied, respectively, 27 mm and 54.5 mm of the 2-h long telophase. On the other hand, the third and last stage in phragmoplast formation covered both the final 35 mm of mitosis and the 6 initial mm of the G1 of the next interphase. A very short (less than 4 mm) stage of microtubular nucleation around the nuclear envelope took place immediately afterwards, before the cortical array of microtubules appeared. The microtubule cycle related to nuclear division started with the apparent activation of the future spindle poles 7.4 mm before prophase was over. The mitotic spindle developed in the 5.6 mm long prometaphase. The spindle functioned in metaphase for the 42 mm it lasted, half spindles being separated for the 37 mm anaphase occupied in these cells.

Wolbachia-induced delay of paternal chromatin condensation does not prevent maternal chromosomes from entering anaphase in incompatible crosses of Drosophila simulans.

The behavior of parental chromosomes during the first mitosis of Drosophila simulans zygotes obtained from unidirectional incompatible crosses is described and it is demonstrated that the condensation of parental chromatin complements was asynchronous. The timing of paternal chromatin condensation appeared to be delayed in these embryos, so that condensed maternal chromosomes and entangled prophase-like paternal fibers congressed in the equatorial plane of the first metaphase spindle. At anaphase the maternal chromosomes migrated to opposite poles of the spindle, whereas the paternal chromatin lagged in the midzone of the spindle. This resulted in dramatic errors in paternal chromatin inheritance leading to the formation of embryos with aneuploid or haploid nuclei. These observations suggest that the anaphase onset of maternal chromosomes is unaffected by the improper alignment of the paternal complement. Since the first metaphase spindle of the Drosophila zygote consists of twin bundles of microtubules each holding one parental complement, we suspect that each half spindle regulates the timing of anaphase onset of its own chromosome set. In normal developing embryos, the fidelity of chromosome transmission is presumably ensured by the relative timing required to prepare parental complements for the orderly segregation that occurs during the metaphase-anaphase transition.

Anaphase in vitro.

Despite their varied appearances, all mitotic spindles have a similar architecture: they are organized as two half spindles, and the microtubules in each half spindle are of the same polarity, with many of the plus ends of the microtubules that grow out from the poles interacting with chromosomes or the microtubules of the other half spindle. During anaphase, the chromosomes move to the spindle poles as the kinetochoreattached microtubules depolymerize (anaphase A), and the spindles elongate due to the presence of pushing forces in the spindle midzone and pulling forces acting on the spindle poles (anaphase B). Although Mazia and Dan (1) first isolated the mitotic apparatus from sea urchin zygotes over 40 years ago, still left unanswered are many questions regarding the identities, locations, and rearrangements of the structural, mechanochemical, and regulatory molecules involved in chromosome separation. Even with a plethora of genetic mutants in various organisms, to determine the nature of these mutants and the relative roles of the affected gene products during mitosis, it is essential to have functional in vitro model systems that can be used to dissect and then reconstitute those elements of the spindle responsible for chromosome movement. The mitotic spindle has many unique features as an organelle that make it difficult to analyze with biochemical techniques. First, the spindle is a dynamic structure; the assembly and disassembly of microtubules contributes to the force generation mechanism responsible for chromosome separation (2). As an analysis of the history of mitosis in vitro shows, media that stabilize spindle structure against the rigors of organelle isolation often inactivate the motile apparatus and render an analysis of the role of microtubule dynamics moot (3). A second problem is that multiple mechanisms of force generation contribute to chromosome separation. Furthermore, redundant motor proteins may participate in any one mechanism of force generation. This has come as a shock to many cell biologists, especially those who tried to explain how chromosomes move by evoking just one mechanochemical system as responsible for all aspects of chromosome movement (reviewed in ref. 4). The current inventory of microtubule motors in the cell is very large, and many of them appear to contribute toward driving the assembly of the spindle apparatus and the subsequent chromosome movement it supports. Thus, spindles are much more complex mechanically than we ever suspected, making a biochemical dissection of spindle function difficult.

Fertilization inDrosophila melanogaster:Centrosome Inheritance and Organization of the First Mitotic Spindle

Microtubule, chromatin, centrosome, and nuclear envelope configurations during the first division of theDrosophila melanogasterzygote were analyzed in order to investigate the organization of the first cleavage spindle and the origin of the functional centrosome. After pronuclear apposition the parental complements congress at the equatorial plane of the metaphase spindle. The chromatids, however, seem to move to the poles in two separate groups in each half spindle, mingling together during telophase, before the formation of the daughter nuclei. The spatial separation of parental complements during the first mitosis is also supported by the behavior of the nuclear envelope of female and male pronuclei. A low frequency of polyspermy is also observed during fertilization inD. melanogaster.

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte: (meiosis/maturation/immunofluorescence/microtubule/spindle).

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.

Artificial Parthenogenesis in Starfish Eggs: Behavior of Nuclei and Chromosomes Resulting in Tetraploidy of Parthenogenotes Produced by the Suppression of Polar Body Extrusion

Artificial parthenogenesis in starfish requires the activation of oocytes and the suppression of the polar body (PB) extrusion. To induce parthenogenesis we have employed a combination of calcium ionophore A23187 for activating oocytes and cytochalasin B (CB) for the suppression of PB extrusions. The treatment of activated oocytes with CB during meiosis I produced eggs lacking polar bodies, i.e., 0pb eggs, and treatment during meiosis II induced eggs bearing the first PB only, i.e., 1pb eggs. About 90% of both 0pb and 1pb eggs developed parthenogenetically with strong synchrony of cell division among eggs. After meiotic chromosomes in CB-treated oocytes separated finally into monads, they came together to form a single nucleus. The nucleus was found to be tetraploid in the 0pb eggs and diploid in the 1pb eggs. Regardless of the difference in ploidy, both 0pb and 1pb eggs developed as tetraploid embryos. Observations of the behavior of meiotic chromosomes and nuclei revealed that in the 0pb eggs, the first round of chromosomal replication was followed by the first cleavage, as in normally fertilized eggs. In the 1pb eggs, on the other hand, two rounds of chromosomal replication were found to precede the first cleavage, indicating the occurrence of one (first) round of chromosomal replication that is not accompanied with cytokinesis. In the first round of mitosis, a bipolar spindle did not appear, but only a half spindle was formed, resulting in the failure of both karyokinesis and cytokinesis. Thus, both types of parthenogenetic eggs became tetraploid before the first cleavage. At the first cleavage, both types of eggs formed a bipolar spindle and divided into a pair of blastomeres. Based on these observations, we suggest that the meiotic centrosomes remaining in these eggs by the failure of PB extrusion are diverted into mitosis-organizing centers in the mitotic spindle, and this results in parthenogenetic development.

Immunofluorescence study of spindle microtubule arrangements during differential gonial mitosis of Acricotopus lucidus (Diptera, Chironomidae).

The changes in microtubule arrangements correlated with the behavior of the germ line limited and soma chromosomes were studied during the last unequal gonial mitosis, the so-called differential mitosis, of the chironomid Acricotopus lucidus by indirect immunofluorescence using a monoclonal anti-beta-tubulin antibody, and by simultaneous staining with the DNA-specific fluorescence dye DAPI. An impressive difference in microtubule density between both half spindles was determined in metaphase and during the monopolar anaphasic migration of the germ line limited chromosomes. In the following normal separation of the soma chromosomes, a similar microtubule density in both half spindles occurred. In each of the half spindles, chromosome movement and spindle elongation occurred independently, and in one half spindle two anaphasic chromosome movements ran off one after another, the second without a simultaneous spindle elongation.

Dithiothreitol prevents membrane fusion but not centrosome or microtubule organization during the first cell cycles in sea urchins.

Dithiothreitol (DTT), a disulfide reducing agent, inhibits the fusion of male and female pronuclei within the activated cytoplasm of sea urchin eggs. The migrations of the pronuclei are not affected by DTT, indicating that microtubule function is not impaired. Centrosomal antigens are detected in the sperm aster and in all subsequent microtubule-based configurations. Nuclear membranes never fuse and the chromatin of male and female pronuclei never mix in the DTT-treated cells. During prophase, when nuclear envelopes break down to undergo mitosis, both sets of chromosomes undergo condensation cycles independent from each other. Both pronuclei initially stain for centrosomal material and surrounding microtubules. With time, the female's centrosomal material as well as the microtubules disappear while the male forms a bipolar spindle. Interestingly, one pole of the paternal mitotic apparatus communicates with the separate maternal chromatin, forming a half spindle which moves the egg-derived chromatin towards its pole. At the time for cell division, the individual karyomeres are not able to fuse their nuclear membranes to reconstitute the blastomere nuclei. When DTT is applied at prometaphase of the first cell cycle, the chromosome cycle continues until next metaphase. Centrosomes also continue their cycle and undergo somewhat atypical splitting during the time for second telophase. Division furrows are initiated but aborted. These results support the hypothesis that disulfide groups are required for membrane fusion of the pronuclei, for membrane fusion of the karyomeres, and for the completion of the division furrow to achieve successful cell division.

Studies on the Sporogenous Lineage in the Moss Timmiella barbuloides

AbstractThis correlated immunofluorescence and electron microscope study reveals that the microtubule arrays during meiosis in Timmiella barbuloides mirror those in other mosses but the organization of the metaphase I spindle is quite different. In other mosses the sagittiform metaphase I spindle initially contains four bands of microtubules derived from the tetrahedral system present at prophase. These bands converge towards the division axis and each half spindle contains two focal points of microtubules straddling a cleavage furrow. In Timmiella the sagittiform spindle also contains four microtubular foci derived from the preprophasic tetrahedron. However, one of these contributes to one half spindle, the other half deriving from the three remaining foci orientated at approximately 120° to each other. In contrast to other mosses the sporocytes in Timmiella are hardly lobed, the cleavage‐furrows ill‐defined, the prophasic plastid positioning in the lobes is also more variable and the organelle band in meiocytes comprises mitochondria alone.

Timing the phases of the microtubule cycles involved in cytoplasmic and nuclear divisions in cells of undisturbed onion root meristems

The duration of the different phases of the microtubule and chromosome cycles were estimated in the native diploid cell populations of Allium cepa L root meristems proliferating undisturbed, under steady state conditions, at the physiological temperature of 15°C. The cycles were coupled by considering their fitting in relation to the short process of nuclear envelope breakdown. In the cycle related to cytoplasmic division, the preprophase band which predicts the future position of the phragmoplast made its appearance, as a wide band, 16 mm before the G 2 to prophase transition, ie it was only present during the final 5% of the total G 2 timing (5 h 30 mm). The band became narrow only 6 mm after prophase had started and it was present in this form for the remaining prophase time (2 h 24 mm). Its disappearance occurred strictly coinciding with nuclear envelope breakdown, at the end of prophase. No microtubules related to cytoplasmic division were apparent until 9 mm after telophase had initiated. The two initial stages of phragmoplast formation which followed occupied, respectively, 27 mm and 54.5 mm of the 2-h long telophase. On the other hand, the third and last stage in phragmoplast formation covered both the final 35 mm of mitosis and the 6 initial mm of the G 1 of the next interphase. A very short (less than 4 mm) stage of microtubular nucleation around the nuclear envelope took place immediately afterwards, before the cortical array of microtubules appeared. The microtubule cycle related to nuclear division started with the apparent activation of the future spindle poles 7.4 mm before prophase was over. The mitotic spindle developed in the 5.6 mm long prometaphase. The spindle functioned in metaphase for the 42 mm it lasted, half spindles being separated for the 37 mm anaphase occupied in these cells.

Anaphase chromosome separation in dividing generative cells ofTradescantia

In dividing generative cells ofTradescantia, kinetochore pairs do not line up on a typical metaphase plate, but instead are distributed along the length and depth of the cell prior to anaphase onset. Kinetochore (K) fibers are linked to each other and to a system of axial microtubule (Mt) bundles in an arrangement that makes discrete half spindles, if present, not immediately obvious. Because such arrangements may have important implications for the conduct of the remainder of division, anaphase events were closely scrutinized using a combination of tubulin and kinetochore immunocytochemistry (the latter with CREST serum). Anaphase appears to consist of three principal processes. Around the time of anaphase onset, K-fibers and surrounding Mts become reorganized into two large superbundles. To each superbundle is attached a set of nonfilial kinetochores bound for one end of the cell. The K-fibers then appear to shorten to varying degrees; in many cases, kinetochores become linked directly to the superbundles. The superbundles then separate in an anaphase B-like process, further moving the kinetochores toward opposite ends of the cell. The superbundles themselves shorten, and distances within the bundles also decrease, such that the kinetochores cluster closer together. The results indicate that reorganization of Mts into superbundles (and the consequential manifestation of bipolarity) is important for orderly chromosome separation.

Meiosis, Spindle Pole Body Cycle and Basidium Ontogeny in the Heterobasidiomycete Agaricostilbum pulcherrimum

Summary Meiosis, the meiotic spindle pole body cycle and aspects of basidial and basidiospore development of the heterobasidiomycete Agaricostilbum pulcherrimum were studied electron microscopically. The extranuclear interphase and prophase spindle pole body consists of a large middle piece and two lateral, multilayered discs. In late prophase I the spindle pole body sinks into a close-fitting depression in the nuclear envelope and becomes perpendicular to the long axis of the nucleus. During the transition from prophase I to prometaphase I, the spindle pole body enters the nucleus and the discs become positioned at the ends of a bipolar spindle. In metaphase-anaphase I and II, the spindle apparatus is entirely intranuclear. During post-telophase I to interphase I, a new spindle pole body appears on the nuclear face of the original disc, extending at an acute angle from the thin electron-dense layer of the original disc. During early meiosis II, the two discs of a nucleus are unequal in size. In prometaphase II the two intranuclear discs lie on the same side of the nucleus and generate two nearly parallel half spindles. In late telophase II/early interphase II each nucleus has an extranuclear spindle pole body with a spherical and a discoidal component. The spherical component forms a new spindle pole body with two discs at interphase IL The ultrastructural characters of nuclear division and the spindle pole body cycle show unique features. The phylogenetic position of Agaricostilbum pulcherrimum is discussed with special reference to meiotic nuclear division.

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.

Kinesin-related cut7 protein associates with mitotic and meiotic spindles in fission yeast.

Several mitotic and meiotic gene products are related to the microtubule motor kinesin, providing insight into the molecular basis of the complex motile events responsible for spindle formation and function. Of these genes, three have been shown to affect spindle structure when mutated. The most severe phenotype is seen in Aspergillus nidulans bimC and Schizosaccharomyces pombe cut7 mutants. In both fungi the intranuclear spindle is bipolar, with microtubules that emanate from spindle pole bodies at either pole, interdigitating in a central overlap zone. In bimC and cut7 mutants, microtubule interdigitation does not appear to take place, instead two unconnected half spindles form and chromosome separation fails. Here we report that cut7 protein concentrates on or near the spindle pole bodies throughout mitotic and meiotic nuclear division and associates with mitotic spindle microtubules in a stage-specific manner, associating with the mid-anaphase B midzone. In cut7ts mutants, spindle pole bodies stain but mitotic microtubules do not.

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.