The impact of chromosomes and centrosomes on spindle assembly as observed in living cells

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video- enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.     

Mitosis in Barbulanympha. II. Dynamics of a two-stage anaphase, nuclear morphogenesis, and cytokinesis

Anaphase in Barbulanympha proceeds in two discrete steps. In anaphase- A, chromosomal spindle fibers shorten and chromosomes move to the stationary centrosomes. In anaphase-B, the central spindle elongates and ("telophasic") bouquets of chromosomes, with kinetochores still connected by the shortened chromosomal fibers to the centrosomes, are moved far apart. The length, width, and birefringence of the central spindle remain unchanged throughout anaphase-A. In anaphase-B, the central spindle elongates up to fivefold. During elongation, the peripheral fibers of the central spindle splay, first anteriorly and then laterally. The remaining central spindle progressively becomes thinner and the retardation decreases; however, the coefficient of birefringence stays approximately constant. The nuclear envelope persists throughout mitosis in Barbulanympha and the nucleus undergoes an intricate morphological change. In prophase, the nucleus engulfs the spindle; in early anaphase-A, the nuclear envelope forms a seam anterior to the spindle, the nucleus thus transforms into a complete sleeve surrounding the central spindle. In late anaphase-A, the middle of the seam opens up in a cleft as the lips part; in anaphase-B, the cleft expands posteriorly, progressively exposing the central spindle. Finally, the cleft partitions the nucleus into two. The nuclear envelope shows an apparent elasticity and two-dimensional fluidity. Localized, transient deformations of the nuclear envelope indicate poleward and counter-poleward forces acting on the kinetochores embedded in the envelope. These forces appear responsible for nuclear morphogenesis as well as anaphase chromosome movement. At the end of anaphase-B, the two rostrate Barbulanympha may swim apart of be poked apart into two daughter cells by another organism cohabiting the host's hindgut.     

Plant TPX2 and related proteins

At the onset of mitosis, microtubules form a bipolar spindle around the prophase nucleus. TPX2 is phosphorylated during mitosis and acts as a spindle assembly factor that nucleates microtubules in the close vicinity of chromosomes, independent of the centrosomes. Furthermore, it activates the kinase Aurora A and targets the Xenopus kinesin-like protein 2 to spindle poles. We have characterized the plant orthologue of TPX2 that possesses all identified functional domains of its animal counterpart. Moreover, we have demonstrated that it is exported before nuclear envelope breakdown and that its activity around the nuclear envelope is essential for prospindle assembly. Here, we compare the sequences of several characterized TPX2 domains, allowing us to define TPX2. We propose that true TPX2 orthologues share simultaneously all these conserved domains and that other proteins possessing only some of these functional blocks may be considered as TPX2-related proteins.Key words: mitosis, microtubules, spindle assembly, TPX2 signature, targeting domains, Prosite motifs, evolution     

The relationship between nuclear envelope-derived annulate lamellae and the asymmetric division of primary spermatocytes in aphids

The structure of dividing primary spermatocytes of Amphorophora tuberculata (Aphididae, Hemiptera) as determined by electron microscopy and serial sectioning is described. The developmental stages examined extend from late prophase I to late telophase I. We looked for any asymmetric organization that could be causally linked to the differences in chromatin behaviour between the two daughter nuclei towards the end of meiosis I of this species. In late prophase I, evaginations of the nuclear envelope in the vicinity of two neigh-bouring centrosomes develop into closed cytoplasmic compartments with a dense content. The compartments open in prometaphase I and come to lie together with fragments of the nuclear envelope within the spindle area. Since nuclear pores are preserved in the membranes, intraspindle annulate lamellae have formed. These and material of presumed nuclear origin associated with them are asymmetrically distributed within the cell. Although dispersed at stages beyond prometaphase I, the material may be largely incorporated into one of the two daughter cells and thus be decisive for further development. Some annulate lamellae form a cap at the chromosome surface opposite to the neighbouring centrosomes in prometaphase I. These membranes may prevent interaction between spindle microtubules and chromosomes until a bipolar spindle forms in metaphase I. At this stage, both the banana-shaped autosomal bivalent and the X univalent occupy the equatorial plane. This is strange, because the X univalent has microtubular connections with one spindle pole and would be expected to migrate towards that pole. Possibly, the kinetochore of the X chromosome is inactive, and remains so in anaphase I, when the X univalent remains located between the two autosomal half-bivalents.M.F. Trendelenburg     

Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes

Chromosome segregation in mammalian oocytes is driven by a microtubule spindle lacking centrosomes. Here, we analyze centrosome-independent spindle assembly by quantitative high-resolution confocal imaging in live maturing mouse oocytes. We show that spindle assembly proceeds by the self-organization of over 80 microtubule organizing centers (MTOCs) that form de novo from a cytoplasmic microtubule network in prophase and that functionally replace centrosomes. Initially distributed throughout the ooplasm, MTOCs congress at the center of the oocyte, where they contribute to a massive, Ran-dependent increase of the number of microtubules after nuclear envelope breakdown and to the individualization of clustered chromosomes. Through progressive MTOC clustering and activation of kinesin-5, the multipolar MTOC aggregate self-organizes into a bipolar intermediate, which then elongates and thereby establishes chromosome biorientation. Finally, a stable barrel-shaped acentrosomal metaphase spindle with oscillating chromosomes and astral-like microtubules forms that surprisingly exhibits key properties of a centrosomal spindle.     

Study of mitosis in root-tip cells ofTriticum turgidum treated with the DNA-intercalating agent ethidium bromide

Summary This work examines mitosis in root-tip cells ofTriticum turgidum treated with the RNA synthesis inhibitor ethidium bromide, using tubulin immunolabeling and electron microscopy. The following aberrations were observed in ethidium bromideaffected cells: (1) incomplete chromatin condensation and nuclear-envelope breakdown; (2) delay of preprophase microtubule band maturation; (3) preprophase microtubule band assembly in cells displaying an interphase appearance of the nucleus; (4) prevention of the prophase spindle formation, caused by inhibition of perinuclear microtubule (Mt) formation and/or inability of the perinuclear Mts to assume bipolarity; (5) organization of an atypical metaphase spindle which is unable to arrange the chromosomes on the equatorial plane; (6) formation of an atypical perinuclear metaphase spindle in cells in which nuclear-envelope breakdown has been almost completely inhibited; (7) inhibition of the anaphase spindle formation as well as of anaphase chromosome movement; (8) disorganization of the atypical mitotic spindle during transition from mitosis to cytokinesis. The observations favor the following hypotheses. Nucleation of prophase spindle Mts is related to the mechanism that causes nuclear-envelope breakdown. The mitotic poles lack Mtnucleating and -organizing properties, and their function does not account for prophase and metaphase spindle assembly. The organization of the prophase spindle is not a prerequisite for the formation of the metaphase spindle; the metaphase spindle seems to be formed de novo by Mts nucleated on the nuclear envelope and/or in the immediate vicinity of chromosomes.Abbreviations 5-AU 5-aminouracil - EB ethidium bromide - EM electron microscopy - k-Mt kinetochore microtubule - Mt microtubule - MTOC microtubule-organizing center - NE nuclear envelope - NEB nuclear-envelope breakdown - PPB preprophase band of microtubules     

The perinuclear microtubule-organizing center and the synaptonemal complex of higher plants share a common antigen: its putative transfer and role in meiotic chromosomal ordering

Recognition of homologous chromosomes during meiotic prophase is associated in most cases with the formation of the synaptonemal complex along the length of the chromosome. Telomeres, located at the nuclear periphery, are preferential initiation sites for the assembly of the synaptonemal complex. In most eukaryotic cells, telomeres cluster in a restricted area, leading to the bouquet configuration in leptotene-zygotene, while this typical organization progressively disappears in late zygotene-pachytene. We wondered whether such striking changes in the intranuclear ordering and pairing of meiotic chromosomes during the progression of prophase I could be correlated with activity of the centrosome and/or microtubule-organizing center (MTOC). Plant cells may be used as a model of special interest for this study as the whole nuclear surface acts as an MTOC, unlike other cell types where MTOCs are restricted to centrosomes or spindle pole bodies. Using a monoclonal antibody (mAb 6C6) raised against isolated calf centrosomes we found that the 6C6 antigen is present over the entire surface of the plant meiotic nucleus, in early prophase I, before chromosomal pairing. At zygotene, short fragments of chromosomes become stained near the nuclear envelope and within the nucleus. At pachytene, after complete synapsis, the labeling specifically concentrates within the synaptonemal complexes, although the nuclear surface is no longer reactive. Ultrastructural localization using immunogold labeling indicates that the 6C6 antigen is colocalized with the synaptonemal complex structures. Later in metaphase I, the antigen is found at the kinetochores. Our data favor the idea that the 6C6 antigen may function as a particular chromosomal passenger-like protein. These observations shed new light on the molecular organization of the plant synaptonemal complex and on the redistribution of cytoskeleton-related antigens during initiation of meiosis. They suggest that antigens of MTOCs are relocated to chromosomes during the synapsis process starting at telomeres and contribute to the spatial arrangement of meiotic chromosomes. Such cytoskeleton-related antigens may acquire different functions depending on their localization, which is cell-cycle regulated.     

Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos

Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition.     

Light and electron microscopy of Rat kangaroo cells in mitosis

Rat kangaroo (PtK₂) cells were fixed and embedded in situ. Cells in mitosis were studied with the light microscope and thin sections examined with the electron microscope. Pericentriolar, osmiophilic material, rather than the centrioles, is probably involved in the formation of astral microtubules during prophase. Centriole migration occurs during prophase and early prometaphase. The nuclear envelope ruptures first in the vicinity of the asters. Nuclear pore complexes disintegrate as envelope fragments are dispersed to the periphery of the mitotic spindle. Microtubules invade the nucleus through gaps of the fragmented envelope. The number of microtubules and the degree of spindle organization increase during prometaphase and are maximal at metaphase. At this stage, chromosomes are aligned on the spindle equator, sister kinetochores facing opposite poles. Cytoplasmic organelles are excluded from the spindle. Prominent bundles of kinetochore microtubules converge towards the poles. Spindles in cold-treated cells consist almost exclusively of kinetochore tubules. Separating daughter chromosomes in early anaphase are connected by chromatin strands, possibly reflecting the rupturing of fibrous connections occasionally observed between sister chromatids in prometaphase. Breakdown of the spindle progresses from late anaphase to telophase, except for the stem bodies. Chromosomes decondense to form two masses. Nuclear envelope reconstruction, probably involving endoplasmic reticulum, begins on the lateral faces. Nuclear pores reappear on membrane segments in contact with chromatin. Microtubules are absent from reconstructed daughter nuclei.This report is to a large part based on a dissertation submitted by the author to the Graduate Council of the University of Florida in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Contribution of Noncentrosomal Microtubules to Spindle Assembly in Drosophila Spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

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, fiuorescent-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 M 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 M 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.     

Sheathed nuclear division in primary spermatocytes of Orgyia antiqua (Lepidoptera, Insecta)

The restructuring of spermatocytes during the first meiotic division is examined in the moth Orgyia antiqua (Lymantriidae, Lepidoptera) using transmission electron microscopy. Particular emphasis was placed on the behaviour of the perispindle membrane system. These membranes develop from layers of smooth endoplasmic reticulum wrapped around the prophase I nucleus and are retained until early telophase I. The original nuclear envelope is dissolved in metaphase I. Polar fenestrae in the perispindle membrane stacks are filled with numerous irregular membrane elements. The formation of new nuclear envelopes around the daughter nuclei takes place inside the perispindle membrane system. Finally, the membrane stacks rupture concomitantly with spindle elongation in late telophase I. Thus, division of primary spermatocytes in Orgyia antiqua has a surprising degree of similarity with the so-called closed mitosis. This mode of division is typical for many protozoa, algae and fungi. In the pertinent cells, the original nuclear envelope persists around the spindle area during nuclear division. In order to distinguish the closed mitosis from the situation in Orgyia antiqua spermatocytes, the term 'sheathed nuclear division' is suggested for the latter.     

TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.     

Origin of the mitotic spindle in onion root cells

Summary Immunofluorescence studies on microtubule arrangement during the transition from prophase to metaphase in onion root cells are presented. The prophase spindle observed at late preprophase and prophase is composed of microtubules converged at two poles near the nuclear envelope; thin bundles of microtubules are tracable along the nuclear envelope. Prior to nuclear envelope breakdown diffuse tubulin staining occurs within the prophase nuclei. During nuclear envelope breakdown the prophase spindle is no longer identifiable and prominent tubulin staining occurs among the prometaphase chromosomes. Patches of condensed tubulin staining are observed in the vicinity of kinetochores. At advanced prometaphase kinetochore bundles of microtubules are present in some kinetochore regions. At metaphase the mitotic spindle is mainly composed of kinetochore bundles of microtubules; pole-to-pole bundles are scarce. Our observations suggest that the prophase spindle is decomposed at the time of nuclear envelope breakdown and that the metaphase spindle is assembled at prometaphase, with the help of kinetochore nucleating action.     

Spindles and centrosomes during male meiosis in Drosophila melanogaster

We have studied the spatial distribution of chromosomes, spindle fibers and centrosomes throughout the first meiotic division in males of Drosophila melanogaster. There seem to be two different types of spindle fibers: those which connect the poles to the chromosomes, and others arranged as cup-shaped hemispheres that reach from the poles to an unstained area on the equator of the cell. These pole-equator fibers could be responsible for positioning the nucleus and distributing cytoplasmic organelles around the nucleus during prophase, so that after meiosis, the daughter cells are provided with equal amounts of preorganized cytoplasmic organelles. These fibers remain until after the daughter nuclei have formed during telophase. An antigen associated with the centrosomes of mitotic spindles appears during meiosis as dispersed particles surrounding the nucleus; these particles might provide the developing spermatids with microtubule-organizing centers.     

Spindle assembly and cytokinesis in the absence of chromosomes during Drosophila male meiosis

A large body of work indicates that chromosomes play a key role in the assembly of both a centrosomal and centrosome-containing spindles. In animal systems, the absence of chromosomes either prevents spindle formation or allows the assembly of a metaphase-like spindle that fails to evolve into an ana-telophase spindle. Here, we show that Drosophila secondary spermatocytes can assemble morphologically normal spindles in the absence of chromosomes. The Drosophila mutants fusolo and solofuso are severely defective in chromosome segregation and produce secondary spermatocytes that are devoid of chromosomes. The centrosomes of these anucleated cells form robust asters that give rise to bipolar spindles that undergo the same ana-telophase morphological transformations that characterize normal spindles. The cells containing chromosome-free spindles are also able to assemble regular cytokinetic structures and cleave normally. In addition, chromosome-free spindles normally accumulate the Aurora B kinase at their midzones. This suggests that the association of Aurora B with chromosomes is not a prerequisite for its accumulation at the central spindle, or for its function during cytokinesis.     

Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

Immunostaining of spindle components in tipulid spermatocytes using a serum against pericentriolar material

First meiotic division of tipulid (Pales ferruginea) spermatocytes was investigated by double immunostaining with anti-tubulin IgG and scleroderma 5051 serum against pericentriolar material (PCM). PCM-like material became visible in late diakinesis in centrosomal areas as well as in kinetochores. Anti-PCM fluorescence was most pronounced in metaphase and diminished again in anaphase. Displacement of one of the centrosomes from the nucleus at diakinesis in Pales spermatocytes leads to the formation of a bipolar, normally functioning spindle possessing one aster and centriole-free spindle pole (AFP). In about 80% of the AFPs observed there were no traces of anti-PCM staining detectable. This finding supports the assumption based on previous studies that polar PCM is not obligatory for spindle pole formation. The chromosomes seem to be able to induce the organization of a half-spindle. The strong anti-PCM fluorescence of the kinetochores observed here may be taken as further indication of tipulid chromosome autonomy regarding spindle formation.