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The mitotic spindle is a complex three-dimensional (3D) apparatus that functions to ensure the faithful segregation of chromosomes during cell division. Our current understanding of spindle architecture is mainly based on a plethora of information derived from light microscopy with rather few insights about spindle ultrastructure obtained from electron microscopy. In this Review, we will provide insights into the history of imaging of mitotic spindles and highlight recent technological advances in electron tomography and data processing, which have delivered detailed 3D reconstructions of mitotic spindles in the early embryo of the nematode Caenorhabditis elegans Tomographic reconstructions provide novel views on spindles and will enable us to revisit and address long-standing questions in the field of mitosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.

Proper positioning of the cleavage furrow is essential for successful cell division. The mitotic spindle, which consists of dynamic astral microtubules and stable equatorial microtubules is responsible for this process. However, little is known about how microtubules are regulated in a time- and region-dependent manner. Here, we show that α-actinin-regulated cortical actin filament integrity is crucial to specify different populations of microtubules during cell division in mammalian cells. Depletion of α-actinin caused aberrant recruitment of centralspindlin, but not aurora B or PRC1, to the tips of astral microtubules, leading to a stable association of astral microtubules with the cortex and induction of ectopic furrowing. Depletion of α-actinin also caused impaired assembly of midzone microtubules, leading to a failure of relocation of aurora B to midzone. Our findings unveil an unexpected yet crucial role for an actin crosslinking protein in the regulation of the localization of the microtubule-associated cytokinetic regulator.

Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression.

We investigated the role of Pav-KLP, a kinesin-6, in the coordination of spindle and cortical dynamics during mitosis in Drosophila embryos. In vitro, Pav-KLP behaves as a dimer. In vivo, it localizes to mitotic spindles and furrows. Inhibition of Pav-KLP causes defects in both spindle dynamics and furrow ingression, as well as causing changes in the distribution of actin and vesicles. Thus, Pav-KLP stabilizes the spindle by crosslinking interpolar microtubule bundles and contributes to actin furrow formation possibly by transporting membrane vesicles, actin and/or actin regulatory molecules along astral microtubules. Modeling suggests that furrow ingression during cellularization depends on: (1) a Pav-KLP-dependent force driving an initial slow stage of ingression; and (2) the subsequent Pav-KLP-driven transport of actin- and membrane-containing vesicles to the furrow during a fast stage of ingression. We hypothesize that Pav-KLP is a multifunctional mitotic motor that contributes both to bundling of interpolar microtubules, thus stabilizing the spindle, and to a biphasic mechanism of furrow ingression by pulling down the furrow and transporting vesicles that deliver new material to the descending furrow.

The formation of the mitotic spindle is controlled by the microtubule organizing activity of the centrosomes and by the effects of chromatin-associated Ran-GTP on the activities of spindle assembly factors. In this study we show that Mars, a Drosophila protein with sequence similarity to vertebrate hepatoma upregulated protein (HURP), is required for the attachment of the centrosome to the mitotic spindle. More than 80% of embryos derived from mars mutant females do not develop properly due to severe mitotic defects during the rapid nuclear divisions in early embryogenesis. Centrosomes frequently detach from spindles and from the nuclear envelope and nucleate astral microtubules in ectopic positions. Consistent with its function in spindle organization, Mars localizes to nuclei in interphase and associates with the mitotic spindle, in particular with the spindle poles, during mitosis. We propose that Mars is an important linker between the spindle and the centrosomes that is required for proper spindle organization during the rapid mitotic cycles in early embryogenesis.

The nuclear mitotic apparatus (NuMA) protein is a component of the nuclear matrix in interphase cells and an essential protein for the formation of mitotic spindle poles. We used herpes simplex virus (HSV), an enveloped DNA virus that replicates in the nucleus, to study the intra-nuclear dynamics of NuMA in infected cells. This study shows that NuMA is extensively modified following HSV infection, including phosphorylation of an unidentified site(s), and that it depends to an extent on viral DNA synthesis. Although NuMA is insoluble in uninfected interphase cells, HSV infection induced solubilisation and dynamic relocalisation of NuMA, whereupon the protein became excluded from viral replication compartments -- sites of virus transcription and replication. Live cell, confocal imaging showed that NuMA localisation dramatically changed from the early stages (diffusely nuclear, excluding nucleoli) to late stages of infection (central diminuition, but remaining near the inner nuclear peripheries). In addition, NuMA knockdown using siRNA suggested that NuMA is important for efficient viral growth. In summary, we suggest that NuMA is required for efficient HSV infection, and identify further areas of research that address how the virus challenges host cell barriers.

Assembly of the mitotic spindle requires a global change in the activity and constitution of the microtubule-binding-protein array at mitotic onset. An important subset of mitotic microtubule-binding proteins localises to the nucleus in interphase and essentially contributes to spindle formation and function after nuclear envelope breakdown. Here, we used a proteomic approach to selectively identify proteins of this category and revealed 50 poorly characterised human gene products, among them the echinoderm microtubule-associated-protein-like gene product, EML3. Indirect immunofluorescence showed that EML3 colocalises with spindle microtubules throughout all mitotic stages. In interphase, EML3 colocalised with cytoplasmic microtubules and accumulated in interphase nuclei. Using YFP-fusion constructs of EML3, we located a nuclear localisation signal and confirmed the microtubule-binding domain of EML3. Functional analysis of EML3 using time-lapse fluorescence microscopy and detailed end-point analysis of phenotypes after siRNA knockdown demonstrates an important role for EML3 in correct metaphase chromosome alignment. Our proteomic identification screen combined with sensitive phenotypic analysis therefore provides a reliable platform for the identification and characterisation of proteins important for correct cell division.

Classical experimental studies on echinoderm zygotes concluded that the juxtaposition of two astral microtubule arrays localizes the stimulus for cytokinetic furrowing. However, recent experimental and genetic studies in Caenorhabditis elegans, Drosophila and mammalian cultured cells implicate microtubules of the central spindle, and regulatory proteins associated with this structure, suggesting that the essential conditions for furrow induction may differ from one animal cell to another. We used micromanipulation and laser microsurgery to create, in three ways, the juxtaposition of astral microtubules in C. elegans embryonic cells. In toroidal cells we observe that furrows initiate both where astral microtubule arrays are juxtaposed, and where the cortex most closely approaches the central spindle. We find that binucleate cells successfully furrow not only across the spindles, but also between unconnected spindle poles. Finally, we find that anucleate cells containing only a pair of centrosomes nevertheless attempt to cleave. Therefore, in C. elegans embryonic cells, as in echinoderms, juxtaposition of two asters suffices to induce furrowing, and neither the chromatin nor the physical structure of the central spindle are indispensable for furrow initiation. However, furrows that cross a central spindle are more likely to complete than those that do not.

We identified an essential kinetochore protein, Mitch, from a genetic screen in D. melanogaster. Mitch localizes to the kinetochore, and its targeting is independent of microtubules (MTs) and several other known kinetochore components. Animals carrying mutations in mitch die as late third-instar larvae; mitotic neuroblasts in larval brains exhibit high levels of aneuploidy. Analysis of fixed D. melanogaster brains and mitch RNAi in cultured cells, as well as video recordings of cultured mitch mutant neuroblasts, reveal that chromosome alignment in mitch mutants is compromised during spindle formation, with many chromosomes displaying persistent mono-orientation. These misalignments lead to aneuploidy during anaphase. Mutations in mitch also disrupt chromosome behavior during both meiotic divisions in spermatocytes: the entire chromosome complement often moves to only one spindle pole. Mutant mitotic cells exhibit contradictory behavior with respect to the spindle assembly checkpoint (SAC). Anaphase onset is delayed in untreated cells, probably because incorrect kinetochore attachment maintains the SAC. However, mutant brain cells and mitch RNAi cells treated with MT poisons prematurely disjoin their chromatids, and exit mitosis. These data suggest that Mitch participates in SAC signaling that responds specifically to disruptions in spindle microtubule dynamics. The mitch gene corresponds to the transcriptional unit CG7242, and encodes a protein that is a possible ortholog of the Spc24 or Spc25 subunit of the Ndc80 kinetochore complex. Despite the crucial role of Mitch in cell division, the mitch gene has evolved very rapidly among species in the genus Drosophila.

Cytokinesis is precisely controlled in both time and space to ensure equal distribution of the genetic material between daughter cells. Incomplete cytokinesis can be associated with developmental or pathological cell division programs leading to tetraploid progenies. In this study we decipher a new mechanism of incomplete cytokinesis taking place in hepatocytes during post-natal liver growth. This process is initiated in vivo after weaning and is associated with an absence of anaphase cell elongation. In this process, formation of a functional contractile actomyosin ring was never observed; indeed, actin filaments spread out along the cortex were not concentrated to the putative site of furrowing. Recruitment of myosin II to the cortex, controlled by Rho-kinase, was impaired. Astral microtubules failed to contact the equatorial cortex and to deliver their molecular signal, preventing activation of the RhoA pathway. These findings reveal a new developmental cell division program in the liver that prevents cleavage-plane specification.

Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which we identified by mass spectrometry analysis as being titin. The spindle matrix proteins skeletor, megator and chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein.

Evidence from various systems suggests that either asters or the midzone of the mitotic spindle are the predominant determinants of cleavage plane position. Disrupting spindle midzone formation in the one-cell Caenorhabditis elegans embryo, such as by using mutants of the centralspindlin component ZEN-4, prevents completion of cytokinesis but does not inhibit furrowing. However, furrowing is inhibited by the simultaneous depletion of ZEN-4 with either PAR-2 or G alpha, which are required for asymmetric divisions. Through studies of other genes required for the presence of an intact spindle midzone containing microtubule bundles, we found that furrowing failed in the absence of PAR-2 or G alpha only when centralspindlin was absent from the furrow. We also found spindle length or microtubule distribution did not correlate with furrow initiation. We propose that centralspindlin acts redundantly with G alpha to regulate furrow initiation.

Cooperativity is well known to promote the speed of some biochemical reactions by accelerating the activity of enzymes. Recent studies have shown that cooperative interactions also function during the formation of a complex cellular structure, the mitotic spindle. Capture of kinetochores by dynamic astral microtubules was originally proposed as the basis of spindle formation. However, mounting evidence indicates that a more complex series of events occurs. It is now clear that there are multiple microtubule nucleation and capture sites throughout the spindle. Kinetochores, centrosomes and microtubules play multiple roles in establishing connections between spindle components and integrating them into a common structure. These data support a modified search-and-capture model that incorporates additional assembly pathways coordinated by a RanGTP gradient.

The spindle occupies a central position in cell division as it builds up the chromosome-separating machine. Here we analysed the dynamics of spindle formation in acentrosomal plant cells by visualizing microtubules labelled with GFP-EB1, GFP-MAP4 and GFP-alpha-tubulin and chromosomes marked by the vital dye SYTO82. During prophase, few microtubules penetrate the nuclear area, followed by nuclear envelope disintegration. During prometaphase, microtubules invading the nuclear space develop a spindle axis from few bipolar microtubule bundles, which is followed by spindle assembly. Using a novel quantitative kymograph analysis based on Fourier transformation, we measured the microtubule growth trajectories of the entire dynamic metaphase spindle. Microtubules initiating from spindle poles either pass through the metaphase plate to form interpolar microtubule bundles or grow until they reach chromosomes. We also noticed a minor fraction of microtubules growing away from the chromosomes. Microtubules grow at 10 microm/minute both at the spindle equator and at the spindle poles. Photobleached marks created on metaphase and anaphase spindles revealed a poleward tubulin flux. During anaphase, the velocity of tubulin flux (2 microm/minute) equals the speed of chromatid-separation. With these findings we identified spatially coordinated microtubule growth dynamics and microtubule flux-based chromosome-separation as important facets of plant spindle operation.

Apicomplexan parasites divide and replicate through a complex process of internal budding. Daughter cells are preformed within the mother on a cytoskeletal scaffold, endowed with a set of organelles whereby in the final stages the mother disintegrates and is recycled in the emerging daughters. How the cytoskeleton and the various endomembrane systems interact in this dynamic process remains poorly understood at the molecular level. Through a random YFP fusion screen we have identified two Toxoplasma gondii proteins carrying multiple membrane occupation and recognition nexus (MORN) motifs. MORN1 is highly conserved among apicomplexans. MORN1 specifically localizes to ring structures at the apical and posterior end of the inner membrane complex and to the centrocone, a specialized nuclear structure that organizes the mitotic spindle. Time-lapse imaging of tagged MORN1 revealed that these structures are highly dynamic and appear to play a role in nuclear division and daughter cell budding. Overexpression of MORN1 resulted in severe but specific defects in nuclear segregation and daughter cell formation. We hypothesize that MORN1 functions as a linker protein between certain membrane regions and the parasite's cytoskeleton. Our initial biochemical analysis is consistent with this model. Whereas recombinant MORN1 produced in bacteria is soluble, in the parasite MORN1 was associated with the cytoskeleton after detergent extraction.

We provide here the first evidence that a distinct midzone is present in the Drosophila melanogaster female meiosis I spindle. This region has the ability to bind the Pavarotti kinesin-like (PAV-KLP) and Abnormal spindle (Asp) proteins, indicating a correct organization of the central spindle microtubules. We also identified the core component centrosomal protein centrosomin (CNN) at an unexpected site within the anaphase I spindle, indicating a role for CNN during the biogenesis of the female meiotic apparatus. However, there are no apparent defects in the midzone organization of cnn oocytes, whereas defects occur later when the central aster forms. The primary mutant phenotype of cnn oocytes is the failure to form a developed central microtubule organizing center (MTOC), although twin meiosis II spindles usually do form. Thus the central MTOC may not be essential for the formation of the inner poles of twin meiosis II spindles, as generally proposed, but it might be involved in maintaining their proper spacing. We discuss the proposal that, in the presence of a central MTOC, a chromatin-driven mechanism of spindle assembly like that described during meiosis I may control the morphogenesis of the twin meiosis II spindles.

Oocyte meiotic spindles of many species are anastral and lack centrosomes to nucleate microtubules. Assembly of anastral spindles occurs by a pathway that differs from that of most mitotic spindles. Here we analyze assembly of the Drosophila oocyte meiosis I spindle and the role of the Nonclaret disjunctional (Ncd) motor in spindle assembly using wild-type and mutant Ncd fused to GFP. Unexpectedly, we observe motor-associated asters at germinal vesicle breakdown that migrate towards the condensed chromosomes, where they nucleate microtubules at the chromosomes. Newly nucleated microtubules are randomly oriented, then become organized around the bivalent chromosomes. We show that the meiotic spindle forms by lateral associations of microtubule-coated chromosomes into a bipolar spindle. Lateral interactions between microtubule-associated bivalent chromosomes may be mediated by microtubule crosslinking by the Ncd motor, based on analysis of fixed oocytes. We report here that spindle assembly occurs in an ncd mutant defective for microtubule motility, but lateral interactions between microtubule-coated chromosomes are unstable, indicating that Ncd movement along microtubules is needed to stabilize interactions between chromosomes. A more severe ncd mutant that probably lacks ATPase activity prevents formation of lateral interactions between chromosomes and causes defective microtubule elongation. Anastral Drosophila oocyte meiosis I spindle assembly thus involves motor-associated asters to nucleate microtubules and Ncd motor activity to form and stabilize interactions between microtubule-associated chromosomes during the assembly process. This is the first complete account of assembly of an anastral spindle and the specific steps that require Ncd motor activity, revealing new and unexpected features of the process.

The assembly of the mitotic spindle after depletion of the major gamma-tubulin isotype by RNA-mediated interference was assessed in the Drosophila S2 cell line. Depletion of gamma-tubulin had no significant effect on the cytoskeletal microtubules during interphase. However, it promoted an increase in the mitotic index, resulting mainly in monopolar and, to a lesser extent, asymmetrical bipolar prometaphases lacking astral microtubules. This mitotic accumulation coincided with the activation of the mitotic checkpoint. Immunostaining with an anti-Asp antibody revealed that the spindle poles, which were always devoid of gamma-tubulin, were unfocused and organized into sub-spindles. Despite the marked depletion of gamma-tubulin, the pericentriolar proteins CP190 and centrosomin were recruited to the spindle pole(s), where they formed three or four dots, suggesting the presence of several centrioles. Electron microscopic reconstructions demonstrated that most of the monopolar spindles exhibited three or four centrioles, indicating centriole duplication with a failure in the separation process. Most of the centrioles were shortened, suggesting a role for gamma-tubulin in centriole morphogenesis. Moreover, in contrast to metaphases observed in control cells, in which the spindle microtubules radiated from the pericentriolar material, in gamma-tubulin-depleted cells, microtubule assembly still occurred at the poles but involved the elongation of centriolar microtubule triplets. Our results demonstrate that, after depletion of gamma-tubulin, the pericentriolar material is unable to promote efficient microtubule nucleation. They point to an alternative mechanism of centrosomal microtubule assembly that contributes to the formation of abnormal, albeit partially functional, mitotic spindles.

Using a meiosis-specific subtracted cDNA library of Schizosaccharomyces pombe, we identified meu14+ as a gene whose expression is upregulated during meiosis. Transcription of meu14+ is induced abruptly after the cell enters meiosis. Its transcription is dependent on the meiosis-specific transcription factor Mei4. In meu14Delta cells, the segregation and modification of the SPBs (spindle pole bodies) and microtubule elongation during meiosis II were aberrant. Meiotic meu14Delta cells consequently produced a high frequency of abnormal tetranucleate cells harboring aberrant forespore membranes and failed to produce asci. In wild-type cells harboring the integrated meu14+-gfp fusion gene, Meu14-GFP first appeared inside the nuclear region at prophase II, after which it accumulated beside the two SPBs at metaphase II. Thereafter, it formed two ring-shaped structures that surrounded the nucleus at early anaphase II. At post-anaphase II, it disappeared. Meu14-GFP appears to localize at the border of the forespore membrane that later develops into spore walls at the end of sporulation. This was confirmed by coexpressing Spo3-HA, a component of the forespore membrane, with Meu14-GFP. Taken together, we conclude that meu14+ is crucial in meiosis in that it participates in both the nuclear division during meiosis II and the accurate formation of the forespore membrane.

Centrin, a 20 kDa calcium-binding protein, is a constituent of contractile basal body-associated fibers in protists and of various centrosomal structures. A construct inducing centrin RNAi was used to study the effect of centrin deficiency in Chlamydomonas. Transformants contained variable amounts of residual centrin (down to 5% of wild-type) and lacked centrin fibers. They displayed a variable flagellar number phenotype with mostly nonflagellate cells, suggesting that centrin is required for basal body assembly. Furthermore, basal bodies often failed to dock to the plasma membrane and to assemble flagella, and displayed defects in the flagellar root system indicating that centrin deficiency interferes with basal body development. Multiple basal bodies caused the formation of additional microtubular asters, whereas the microtubular cytoskeleton was disordered in most cells without basal bodies. The number of multinucleated cells was increased, indicating that aberrant numbers of basal bodies interfered with the cytokinesis of Chlamydomonas. In contrast to wild-type cells, basal bodies in centrin-RNAi cells were separated from the spindle poles, suggesting a role of centrin in tethering basal bodies to the spindle. To test whether an association with the spindle poles is required for correct basal body segregation, we disrupted centrin fibers in wild-type cells by over-expressing a nonfunctional centrin-GFP. In these cells, basal bodies were disconnected from the spindle but segregation errors were not observed. We propose that basal body segregation in Chlamydomonas depends on an extranuclear array of microtubules independent of the mitotic spindle.

The meiosis-I spindle in flea-beetle spermatocytes is unusual in that the autosomes and univalent sex chromosomes are separated by a mitochondrial sheath and move polewards at different times. To help understand the basis for this interesting chromosome behaviour, and to gather more detailed information about it, we studied microtubule distributions throughout meiosis I using immunofluorescence and confocal microscopy, and took careful measurements of pole and kinetochore positions at all stages of division. Our results show that, by late prophase, there is a spindle-shaped cytoplasmic array of microtubules in the central part of the cell, with the nucleus at the periphery. Following nuclear envelope breakdown, both autosomes and sex chromosomes become associated with cytoplasmic microtubules, although only the autosomes move centrally to the 'cytoplasmic spindle'. The two unpaired sex chromosomes remain at the cell periphery and appear to be connected to each other by a microtubule bundle extending between their kinetochores. These bundles often persist into anaphase. Analysis of measurements taken from fixed/stained cells supports previous observations that sex chromosomes move part way to the pole in early prometaphase and then stop. The measurements also suggest that during autosomal anaphase, spindle elongation precedes autosome movement to the poles and polewards movement of sex chromosomes is limited or absent when autosomes are moving polewards.

Astrin is a mitotic-spindle-associated protein expressed in most human cell lines and tissues. However, its functions in spindle organization and mitosis have not yet been determined. Sequence analysis revealed that astrin has an N-terminal globular domain and an extended coiled-coil domain. Recombinant astrin was purified and characterized by CD spectroscopy and electron microscopy. Astrin showed parallel dimers with head-stalk structures reminiscent of motor proteins, although no sequence similarities to known motor proteins were found. In physiological buffers, astrin dimers oligomerized via their globular head domains and formed aster-like structures. Silencing of astrin in HeLa cells by RNA interference resulted in growth arrest, with formation of multipolar and highly disordered spindles. Chromosomes did not congress to the spindle equator and remained dispersed. Cells depleted of astrin were normal during interphase but were unable to progress through mitosis and finally ended in apoptotic cell death. Possible functions of astrin in mitotic spindle organization are discussed.

Spindle checkpoint proteins, such as Mad2 and BubR1, and the motors dynein/dynactin and CENP-E usually leave kinetochores prior to anaphase onset by microtubule-dependent mechanisms. Likewise, 'chromosome passenger proteins' including INCENP are depleted from the centromeres after anaphase onset and then move to the midzone complex, an event that is essential for cytokinesis. Here we test whether the cell cycle changes that occur at anaphase onset require or contribute to the depletion of kinetochore and centromere proteins independent of microtubules. This required the development of a novel non-antibody method to induce precocious anaphase onset in vivo by using a bacterially expressed fragment of the spindle checkpoint protein Mad1 capable of activating the APC/C, called GST-Mad1F10. By injecting PtK1 cells in nocodazole with GST-Mad1F10 and processing the cells for immunofluorescence microscopy after anaphase sister chromatid separation in nocodazole we found that Mad2, BubR1, cytoplasmic dynein, CENP-E and the 3F3/2 phosphoepitope remain on kinetochores. Thus depletion of these proteins (or phosphoepitope) at kinetochores is not required for anaphase onset and anaphase onset does not produce their depletion independent of microtubules. In contrast, both microtubules and anaphase onset are required for depletion of the 'chromosome passenger' protein INCENP from centromeres, as INCENP does not leave the chromosomes prior to anaphase onset in the presence or absence of microtubules, but does leave the centromeres after anaphase onset in the presence of microtubules.

Although Drosophila larval neuroblasts are routinely used to define mutations affecting mitosis, the dynamics of karyokinesis in this system remain to be described. Here we outline a simple method for the short-term culturing of neuroblasts, from Drosophila third instar larvae, that allows mitosis to be followed by high-resolution multi-mode light microscopy. At 24 degrees C, spindle formation takes 7+/-0.5 minutes. Analysis of neuroblasts containing various GFP-tagged proteins (e.g. histone, fizzy, fizzy-related and alpha-tubulin) reveals that attaching kinetochores exhibit sudden, rapid pole-directed motions and that congressing and metaphase chromosomes do not undergo oscillations. By metaphase, the arms of longer chromosomes can be resolved as two chromatids, and they often extend towards a pole. Anaphase A and B occur concurrently, and during anaphase A chromatids move poleward at 3.2+/-0.1 microm/minute, whereas during anaphase B the spindle poles separate at 1.6+/-01 microm/minute. In larger neuroblasts, the spindle undergoes a sudden shift in position during midanaphase, after which the centrally located centrosome preferentially generates a robust aster and stops moving, even while the spindle continues to elongate. Together these two processes contribute to an asymmetric positioning of the spindle midzone, which, in turn, results in an asymmetric cytokinesis. Bipolar spindles form predominately (83%) in association with the separating centrosomes. However, in 17% of the cells, secondary spindles form around chromosomes without respect to centrosome position: in most cases these spindles coalesce with the primary spindle by anaphase, but in a few they remain separate and define additional ectopic poles.

We examined the relationship between the spindle orientation and the determination site of cytokinesis in brown algal cells using polyspermic zygotes of Scytosiphon lomentaria. When two male gametes fuse with one female gamete, the zygote has two pairs of centrioles derived from male gametes and three chloroplasts from two male and one female gametes. Just before mitosis, two pairs of centrioles duplicate and migrate towards the future mitotic poles. Spindle MTs develop and three or four spindle poles are formed. In a tri-polar spindle, one pair of centrioles shifts away from the spindle, otherwise, two pairs of centrioles exist adjoining at one spindle pole. Chromosomes arrange at several equators of the spindle. As a result of these multipolar mitoses, three or four daughter nuclei developed. Subsequently, these daughter nuclei form a line along the long axis of the cell. Cell partition always takes place between daughter nuclei, perpendicular to the long axis of the cell. Three or four daughter cells are produced by cytokinesis. Some of the daughter cells after cytokinesis do not have a nucleus, but all of them always contain the centrosome and chloroplast. Therefore, the number of daughter cells always coincides with the number of centrosomes or microtubule organizing centers (MTOCs). These results show that the cytokinetic plane in the brown algae is determined by the position of centrosomes after mitosis and is not dependent on the spindle position.

The centrosome consists of two centrioles surrounded by the pericentriolar material (PCM). In late G2 phase, centrosomes enlarge by recruiting extra PCM, and concomitantly its microtubule nucleation activity increases dramatically. The regulatory mechanisms of this dynamic change of centrosomes are not well understood. Protein phosphatase 4 (PP4) is known to localize to mitotic centrosomes in mammals and Drosophila. An involvement of PP4 in the mitotic spindle assembly has been implicated in Drosophila, but in vivo functions of PP4 in other organisms are largely unknown. Here we characterize two Caenorhabditis elegans PP4 genes, named pph-4.1 and pph-4.2. Inhibition of the function of each gene by RNA-mediated interference (RNAi) revealed that PPH-4.1 was essential for embryogenesis but PPH-4.2 was not. More specifically, PPH-4.1 was required for the formation of spindles in mitosis and sperm meiosis. However, this phosphatase was apparently dispensable for female meiotic divisions, which do not depend on centrosomes. In the cell depleted of pph-4.1 activity, localization of gamma-tubulin and a Polo-like kinase homologue to the centrosome was severely disturbed. Immunofluorescence staining revealed that PPH-4.1 was present at centrosomes from prophase to telophase, but not during interphase. These results indicate that PPH-4.1 is a centrosomal protein involved in the recruitment of PCM components to the centrosome, and is essential for the activation of microtubule nucleation potential of the centrosome. Furthermore, chiasmata between homologous chromosomes were often absent in oocytes that lacked pph-4.1 activity. Thus, besides promoting spindle formation, PPH-4.1 appears to play a role in either the establishment or the maintenance of chiasmata during meiotic prophase I.

The tachyzoite stage of the protozoan parasite Toxoplasma gondii has two populations of microtubules: spindle microtubules and subpellicular microtubules. To determine how these two microtubule populations are regulated, we investigated microtubule behavior during the cell cycle following treatment with microtubule-disrupting drugs. Previous work had established that the microtubule populations are individually nucleated by two distinct microtubule-organizing centers (MTOCs): the apical polar ring for the subpellicular microtubules and spindle pole plaques/centrioles for the spindle microtubules. When replicating tachyzoites were treated with 0.5 microM oryzalin or 1.0 mM colchicine they retained the capacity to form a spindle and undergo nuclear division. Although these parasites could complete budding, they lost the bulk of their subpellicular microtubules and the ability to reinvade host cells. Both nascent spindle and subpellicular microtubules were disrupted in 2.5 microM oryzalin or 5.0 mM colchicine. Under these conditions, parasites grew in size and replicated their genome but were incapable of nuclear division. After removal from 0.5 microM oryzalin, Toxoplasma tachyzoites were able to restore normal subpellicular microtubules and a fully invasive phenotype. When oryzalin was removed from Toxoplasma tachyzoites treated with 2.5 microM drug, the parasites attempted to bud as crescent-shaped tachyzoites. Because the polyploid nuclear mass could not be correctly segregated, many daughter parasites lacked nuclei altogether although budding and scission from the maternal mass was able to be completed. Multiple MTOCs permit Toxoplasma tachyzoites to control nuclear division independently from cell polarity and cytokinesis. This unusual situation grants greater cell cycle flexibility to these parasites but abolishes the checks for coregulation of nuclear division and cytokinesis found in other eukaryotes.

Cells of the fission yeast Schizosaccharomyces pombe divide by the contraction of the F-actin ring formed at the medial region of the cell. We investigated the process of F-actin ring formation in detail using optical sectioning and three-dimensional reconstruction fluorescence microscopy. In wild-type cells, formation of an aster-like structure composed of F-actin cables and accumulation of F-actin cables were recognized at the medial cortex of the cell during prophase to metaphase. The formation of the aster-like structure seemed to initiate from branching of the longitudinal F-actin cables at a site near the spindle pole bodies, which had been duplicated but not yet separated. A single cable extended from the aster and encircled the cell at the equator to form a primary F-actin ring during metaphase. During anaphase, the accumulated F-actin cables were linked to the primary F-actin ring, and then all of these structures seemed to be packed to form the F-actin ring. These observations suggest that formation of the aster-like structure and the accumulation of the F-actin cables at the medial region of the cell during metaphase may be required to initiate the F-actin ring formation. In the nda3 mutant, which has a mutation in ss-tubulin and has been thought to be arrested at prophase, an F-actin ring with accumulated F-actin cables similar to that of anaphase wild-type cells was formed at a restrictive temperature. Immediately after shifting to a permissive temperature, this structure changed into a tightly packed ring. This suggests that the F-actin ring formation progresses beyond prophase in the nda3 cells once the cells enter prophase. We further examined F-actin structures in both cdc12 and cdc15 early cytokinesis mutants. As a result, Cdc12 seemed to be required for the primary F-actin ring formation during prophase, whereas Cdc15 may be involved in both packing the F-actin cables to form the F-actin ring and rearrangement of the F-actin after anaphase. In spg1, cdc7 and sid2 septum initiation mutants, the F-actin ring seemed to be formed in order.

GLC7 encodes the catalytic subunit of type 1 protein serine/threonine phosphatase (PP1) in the yeast Saccharomyces cerevisiae. Here we have characterized the temperature-sensitive glc7-10 allele, which displays aberrant bud morphology and an abnormal actin cytoskeleton at the restrictive temperature. At 37 degrees C glc7-10 strains accumulated a high proportion of budded cells with an unmigrated nucleus, duplicated spindle pole bodies, a short spindle, delocalized cortical actin and 2C DNA content, indicating a cell cycle block prior to the metaphase to anaphase transition. glc7-10 was suppressed by growth on high osmolarity medium and exhibited temperature-sensitive cell lysis upon hypo-osmotic stress. Pkc1p, the yeast protein kinase C homolog which is thought to regulate the Mpk1p MAP kinase pathway involved in cell wall remodelling and polarized cell growth, was found to act as a dosage suppressor of glc7-10. Although neither activation of BCK1 (MEKK) by the dominant BCK1-20 mutation nor increased dosage of MKK1 (MEK) or MPK1 (MAP kinase) mimicked PKC1 as a glc7-10 dosage suppressor, extra copies of genes encoding upstream components of the Pkc1p pathway such as ROM2, RHO2, HCS77/WSC1/SLG1 and MID2 also suppressed glc7-10 effectively. Conversely, mpk1delta glc7-10 and bck1delta glc7-10 double mutants displayed a synthetic cell lysis defect compared with each single mutant and glc7-10 was hypersensitive to reduced PKC1 function, displaying highly aberrant morphologies and inviability even at the normally permissive temperature of 26 degrees C. Dephosphorylation by PP1 therefore functions positively to promote cell integrity, bud morphology and polarization of the actin cytoskeleton and glc7-10 cells require higher levels of Pkc1p activity to sustain these functions.

Spindle pole bodies in the fission yeast Schizosaccharomyces pombe are required during meiosis, not only for spindle formation but also for the assembly of forespore membranes. The spo15 mutant is defective in the formation of forespore membranes, which develop into spore envelopes. The spo15(+)gene encodes a protein with a predicted molecular mass of 223 kDa, containing potential coiled-coil regions. The spo15 gene disruptant was not lethal, but was defective in spore formation. Northern and western analyses indicated that spo15(+) was expressed not only in meiotic cells but also in vegetative cells. When the spo15-GFP fusion gene was expressed by the authentic spo15 promoter during vegetative growth and sporulation, the fusion protein colocalized with Sad1p, which is a component of spindle pole bodies. Meiotic divisions proceeded in spo15delta cells with kinetics similar to those in wild-type cells. In addition, the morphology of the mitotic and meiotic spindles and the nuclear segregation were normal in spo15delta. Intriguingly, transformation of spindle pole bodies from a punctate to a crescent form prior to forespore membrane formation was not observed in spo15delta cells. We conclude that Spo15p is associated with spindle pole bodies throughout the life cycle and plays an indispensable role in the initiation of spore membrane formation.

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.

Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.

Cytocentrin is a cytosolic protein that transiently associates with the mitotic spindle poles in early prophase, and dissociates from them after completion of mitosis. Cloning of its cDNA demonstrated a high degree of homology with three proteins known to specifically interact with an activated form of Ral. Herein we demonstrate that overexpression of cytocentrin inhibits assembly of the mitotic spindle without affecting polymerization or distribution of interphase microtubules. Conversely, loss of cytocentrin expression leads to formation of monopolar spindles. These results indicate that association of cytocentrin with the centrosome may be essential for a timely separation of the diplosomes. They also implicate Ral GTPases and their related pathways in the assembly and function of the mitotic apparatus.

The early Caenorhabditis elegans embryo divides with a stereotyped pattern of cleavages to produce cells that vary in developmental potential. Differences in cleavage plane orientation arise between the anterior and posterior cells of the 2-cell embryo as a result of asymmetries in centrosome positioning. Mechanisms that position centrosomes are thought to involve interactions between microtubules and the cortex, however, these mechanisms remain poorly defined. Interestingly, in the early embryo the shape of the centrosome predicts its subsequent movement. We have used rhodamine-tubulin and live imaging techniques to study the development of asymmetries in centrosome morphology and positioning. In contrast to studies using fixed embryos, our images provide a detailed characterization of the dynamics of centrosome flattening. In addition, our observations of centrosome behavior in vivo challenge previous assumptions regarding centrosome separation by illustrating that centrosome flattening and daughter centrosome separation are distinct processes, and by revealing that nascent daughter centrosomes may become separated from the nucleus. Finally, we provide evidence that the midbody specifies a region of the cortex that directs rotational alignment of the centrosome-nucleus complex and that the process is likely to involve multiple interactions between microtubules and the cortex; the process of alignment involves oscillations and overshoots, suggesting a multiplicity of cortical sites that interact with microtubules.

The monoclonal antibody 78H6 recognises an 85 kDa component of the yeast spindle pole body. Here we identify and characterise this component as Spc72p, the product of YAL047C. The sequence of SPC72 contains potential coiled-coil domains; its overexpression induced formation of large polymers that were strictly localised at the outer plaque and at the bridge of the spindle pole body. Immunoelectron microscopy confirmed that Spc72p was a component of these polymers. SPC72 was found to be non-essential for cell growth, but its deletion resulted in abnormal spindle positioning, aberrant nuclear migration and defective mating capability. Precisely, deletion of SPC72 resulted in a decreased number of astral microtubules: early in the cell cycle only few were detectable, and these were unattached to the spindle pole body in small-budded cells. Later in the cell cycle few, if any, remained, and they were unable to align the spindle properly. We conclude that Spc72p is not absolutely required for nucleation per se, but is needed for normal abundance and stability of astral microtubules.

Spindle poles are discernible by light microscopy as the sites where microtubules converge at the ends of both mitotic and meiotic spindles. In most cell types centrosomes are present at spindle poles due to their dominant role in microtubule nucleation. However, in some specialized cell types microtubules converge into spindle poles in the absence of centrosomes. Thus, spindle poles in centrosomal and acentrosomal cell types are structurally different, and it is this structural dichotomy that has created confusion as to the mechanism by which microtubules are organized into spindle poles. This review summarizes a series of recent articles that begin to resolve this confusion by demonstrating that spindle poles are organized through a common mechanism by a conserved group of non-centrosomal proteins in the presence or absence of centrosomes.

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Cdc28 is a cyclin-dependent protein kinase of Saccharomyces cerevisiae that is required for the G1/S and G2/M transitions of the cell division cycle. All previously described cdc28 mutants aside from cdc28-1N arrest division specifically in the G1 phase. cdc28-1N arrests division in G2/mitosis. We show here that the cdc28-109 mutant exhibits a mixed cell division arrest at 37 degrees C with cells in both the G1 and G2 phases. In order to identify proteins that functionally interact with Cdc28, we isolated mutants that are colethal with cdc28-109 at its permissive temperature. We describe here our phenotypic analysis of two such mutants, hsf1-82 and ydj1-10, that affect the heat shock transcription factor and a yeast dnaj-like protein chaperone, respectively. hsf1-82 and ydj1-10 temperature-sensitive mutants arrest the cell division cycle at several stages. However, one predominant class of cells in both mutants was arrested with a large bud and a single vertex of microtubules. Electron microscopic analysis of such hsf1-82 cells showed that they contained an unduplicated spindle pole body with an enlarged half-bridge. Two-dimensional gel electrophoresis of total cell proteins revealed that the hsf1-82 cells were specifically defective in the expression of the Hsc82 and Hsp82 proteins. Furthermore, the hsf1-82 mutation was suppressed by the HSC82 gene on a multicopy plasmid that restored Hsc82 protein to high levels in these cells. These results show that Hsf1 is required for spindle pole body duplication at 37 degrees C.

The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP). In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome. Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Camlp) in fission yeast, where calmodulin had not been localized by any method. We find GFP-Camlp also localizes both to sites of polarized cell growth and to the fission yeast SPB. The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant. Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis. Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed. Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells. Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.

Specific changes in spatial order occur during cell cycle progression in fission yeast. Growth of the rod-shaped cells is highly regulated and undergoes a cell cycle and size-regulated switch from monopolar to bipolar tip extension. During both phases of growth, the interphase nucleus is maintained in a central location. Following the separation of the genome to the cell tips in mitosis, the two nuclei migrate back towards the cell equator before stopping in two new positions that will become the middle of the two new cells. Here we use simultaneous labeling of microtubules, chromatin and spindle pole bodies in wild-type and cdc mutants, to show that nuclear positioning is achieved by regulation of spindle pole body-mediated nuclear migration. We show that the number and location of nuclear positioning signals is regulated in a cell cycle-specific manner and that spindle pole body-mediated forces are likely to be responsible for maintaining correct nuclear position once the nuclei have reached the appropriate position in the cell. Accentuating the movement of the nuclei back towards the cell equator after mitosis by artificially increasing cell length shows that the spindle pole body leads the nucleus during this migration. When multiple spindle pole bodies are associated with the same or different nuclei they all go to the same point indicating that the different spindle pole bodies are responding to the same positional cue. In a septation-defective mutant cell, which contains four nuclei, the spindle pole bodies on the four different nuclei initially group as two pairs in regions that would become the middle of the new cells, were the cell able to divide. In the subsequent interphase, the nuclei aggregate as a group of four in the centre of the cell. The presence of two or three clusters of spindle pole bodies in larger cells with eight nuclei suggests that the mechanisms specifying the normally central location for multiple nuclei may be unable to operate properly as the cells get larger. Perturbation of microtubules with the microtubule poison thiabendazole prevents the spindle pole body clustering in septation mutants, demonstrating that nuclear positioning requires a functional microtubule cytoskeleton.

The nuclear envelope is central to nuclear structure and function. It plays a role in maintaining nuclear shape, allowing the exchange of macromolecules between the nucleus and the cytoplasm (via the nuclear pore complexes), and providing attachment sites for microtubules during chromosome segregation and nuclear migration (via the spindle pole body). We have isolated an essential yeast gene, NNF1 that is required for a number of nuclear functions. Cells depleted of Nnf1p or containing a temperature-sensitive nnf1 mutation have elongated microtubules and become bi- and multinucleate. They also have a fragmented nucleolous and accumulate poly(A)+ RNA inside the nucleus. A similar constellation of phenotypes has been reported in cells carrying mutations in a number of nuclear pore proteins, components of the Ran GTPase cycle, and the nuclear localization sequence receptor protein. Our results suggest that Nnf1p plays a role in a number of nuclear functions.

NuMA is a 236 kDa nuclear protein that is required for the organization of the mitotic spindle. To determine how NuMA redistributes in the cell during mitosis, we have examined the behavior of NuMA in a mammalian mitotic extract under conditions conducive to the reassembly of interphase nuclei. NuMA is a soluble protein in mitotic extracts prepared from synchronized cultured cells, but forms insoluble structures when the extract becomes non-mitotic (as judged by the inactivation of cdc2/cyclin B kinase and the disappearance of mpm-2-reactive antigens). These NuMA-containing structures are irregularly shaped particles of 1-2 microm in diameter and their assembly is specific because other nuclear components such as the lamins remain soluble in the extract under these conditions. NuMA is dephosphorylated during this assembly process, and the assembly of these NuMA-containing structures is catalyzed by protein dephosphorylation because protein kinase inhibitors enhance their formation and protein phosphatase inhibitors block their formation. Finally, immunodepletion demonstrates that NuMA is an essential structural component of these insoluble particles, and electron microscopy shows that the particles are composed of a complex interconnected network of foci. These results demonstrate that phosphorylation regulates the solubility of NuMA in a mammalian mitotic extract, and the spontaneous assembly of NuMA into extensive structures upon dephosphorylation supports the conclusion that NuMA serves a structural function.

We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is temperature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term 'abnormal metaphase'. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with 'broken spindles', where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.

The spindle pole localization of gamma-tubulin was compared in wild type and acentriolar cultured Drosophila cells using polyclonal antibodies specifically raised against the carboxy terminal amino acid sequence of Drosophila gamma-tubulin-1 (-KSEDSRSVTSAGS). During interphase, gamma-tubulin was present in the centrosome of wild type cells and accumulated around this organelle in a cell cycle dependent manner. In contrast, no such structure was observed in acentriolar cells. Wild type mitoses were homogeneously composed of biconical spindles, with two centrosome-associated gamma-tubulin spots at the poles. The mitotic apparatuses observed in the acentriolar cells were heterogeneous; multipolar mitoses, bipolar mitoses with a barrel-shaped spindle and bipolar mitoses with biconical spindles were observed. In acentriolar cells, gamma-tubulin accumulation at mitotic poles was dependent on spindle microtubule integrity. Most acentriolar spindles presented a dispersed gamma-tubulin labeling at the poles. Only well polarized and biconical acentriolar spindles showed a strong gamma-tubulin polar spot. Finally, acentriolar mitotic poles were not organized around true centrosomes. In contrast to wild type cells, in acentriolar cells the Bx63 centrosome-associated antigen was absent and the gamma-tubulin containing material dispersed readily following microtubule disassembly. These observations confirm that gamma-tubulin plays an essential role in the nucleation of microtubules even in the absence of mitotic polar organelles. In addition the data suggest that the mechanisms involved in the bipolarization of wild type and acentriolar mitoses are different, and that centrioles play a role in the spatial organization of the nucleating material containing gamma-tubulin.

To study the role of the metaphase spindle during the period of oocyte activation, mouse oocytes were fertilised or activated parthenogenetically in the presence or absence of the microtubule inhibitor nocodazole. In both cases, nocodazole caused the disappearance of the spindle and prevented the passage of the oocytes into interphase. However, the calcium spiking responses of the oocytes were not affected by nocodazole, being repetitive after fertilisation and a single spike after activation. If, after their activation or fertilisation in nocodazole, oocytes were later removed from the drug, only those that had been fertilised progressed into interphase. This progress was associated with continuing calcium spiking. Moreover, both the spiking and the progress to interphase could be blocked or reduced in incidence by removal of external calcium or addition of 5,5'-dimethyl BAPTA-AM. Oocytes that had been activated by ethanol in the presence of nocodazole and then removed from it, to allow re-formation of the spindle, only progressed into interphase if given a second exposure to ethanol, thereby eliciting a second calcium transient. These results show that exit from meiotic M-phase requires the simultaneous presence of a fully intact spindle during the release of calcium and that those factors leading to the degradation of cyclin B are only activated transiently. Since cyclin is being degraded continuously in the metaphase-II-arrested mouse oocyte and since this degradation is microtubule-dependent, these data suggest that the superimposition of a high concentration of intracellular calcium is required to tilt the equilibrium further in favour of cyclin degradation if exit from M-phase is to occur.

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48-50 kDa in isolated spindles and centrosomes from CHO cells.

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.

Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is t 1/2 = 31 +/- 6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (t 1/2 = 67 +/- seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all four MT arrays are more consistent with subunit exchange by the mechanism of dynamic instability than treadmilling. This is the first quantification of MT dynamics in plant cells.