Direct observation of mitotic spindle elongation in vitro.

Reactivation of spindle elongation in vitro is correlated with the phosphorylation of a 205 kd spindle-associated protein

The role of tubulin polymerization during spindle elongation in vitro.

Because cohesion prevents sister-chromatid separation and spindle elongation, cohesion dissolution may trigger these two events simultaneously. However, the relatively normal spindle elongation kinetics in yeast cohesin mutants indicates an additional mechanism for the temporal control of spindle elongation. Here we show evidence indicating that S-phase CDK (cyclin dependent kinase) negatively regulates spindle elongation. In contrast, mitotic CDK promotes spindle elongation by activating Cdc14 phosphatase, which reverses the protein phosphorylation imposed by S-phase CDK. Our data suggest that S-phase CDK negatively regulates spindle elongation partly through its phosphorylation of a spindle pole body (SPB) protein Spc110. We also show that hyperactive S-phase CDK compromises the microtubule localization of Stu2, a processive microtubule polymerase essential for spindle elongation. Strikingly, we found that hyperactive mitotic CDK induces uncoupled spindle elongation and sister-chromatid separation in securin mutants (pds1Δ), and we speculate that asynchronous chromosome segregation in pds1Δ cells contributes to this phenotype. Therefore, the tight temporal control of spindle elongation and cohesin cleavage assure orchestrated chromosome separation and spindle elongation.

The positions of the two sets of chromosome kinetochores, the spindle poles, cell membrane adjacent to the poles, and cleavage furrow of grasshopper neuroblasts in culture at 38°C were determined at short-time intervals during anaphase. The percent of motion due to poleward movement and spindle elongation, which coincide in time, were calculated for each minute, the former falling from 61% in the first minute to 15% in the seventh minute, and increasing to 86% in the final minute, probably as a result of pressure and bending of the spindle. Of the total chromosome movement during anaphase 44.6% is due to poleward movement of the daughter kinetochores and 55.4% to spindle elongation. The maximum velocity of a set of kinetochores is 3.41 μm/min and the mean velocity 1.86 μm/min (one-half the rate of separation). Various studies of anaphase chromosome movement in different cells and different species suggest certain generalizations, some of which are based on very small samples and so must be considered quite tentative: (1) The combination of poleward movement and spindle elongation is much more frequent than either acting alone. (2) These components of movement may coincide in time, overlap, or spindle elongation may follow poleward movement, but spindle elongation never begins before poleward chromosome movement. (3) There is an optimum temperature for the rate of chromosome movement, above and below which the rate gradually decreases. (4) In homoiothermic animals this optimum occurs at normal body temperature. (5) In homoiothermic animals the velocity falls more rapidly with a decrease in temperature than in poikilothermic animals. (6) Animals with large chromosomes (amphibia, grasshoppers) have higher chromosome velocities than those with small chromosomes. (7) Non-meiotic cells and secondary spermatocytes have higher velocities than primary spermatocytes of the same species. (8) Chromosome velocity is lower in malignant than non-malignant cells. (9) Chromosome velocity tends to be positively correlated with the distance the chromosomes travel during anaphase.

The mitotic spindle in the fission yeast Schizosaccharomyces pombe is a single bundle of microtubules that elongates to segregate the chromosomes during anaphase B. The mechanical properties of the spindle and the forces driving its elongation remain poorly defined. Here, we analyzed how spindles react to mechanical and genetic perturbations to uncover their mechanical properties. Treatment of cells with osmotic oscillations and blue light led to a consistent phenotype of spindle buckling and breakage in mid-anaphase. The stalling of pole separation and reduced rates of spindle elongation indicated that spindles elongate and buckle under increased mechanical load. The structural integrity of the bent spindles was dependent on Ase1 (PRC1), while the spindle elongation rate was dependent on motor proteins Klp9 (kinesin-6) and Cut7 (kinesin-5). Modeling of bent spindle shapes revealed that most spindles behave mechanically as a beam in which the midzone region is one to two times as rigid than the rest of the beam. Upon reaching a threshold size, bent spindles broke at a specific fragile site near the edge of the spindle midzone. Our findings in this simple fission yeast spindle are relevant to the mechanics of more complex metazoan spindles.

Spindle elongation is crucial to normal chromosome separation in eukaryotes; in particular, it is required for or associated with the extension of distance between spindle poles and the further moving apart of the already separated chromosomes. However, little is known about the relationship between spindle elongation and the status of chromosome separation, and it is unknown whether spindle elongation in different organisms shares any quantitative feature. The Arabidopsis ask1-1 mutant might be a unique material for addressing these questions because it appears to have functional spindles, but a severe defect in homolog separation at male anaphase I (M. Yang, Y. Hu, M. Lodhi, W.R. McCombie, H Ma [1999] Proc Natl Acad Sci USA 96: 11416-11421). We have characterized male meiotic spindle lengths in wild-type and the ask1-1 mutant plants. We observed that during meiosis I some ask1-1 cells had spindles that were similar in length to fully elongated normal spindles, but the chromosomes in these cells did not show appreciable movement from the equator. Furthermore, greater movement of chromosomes from the equator was usually found in the ask1-1 cells that had longer than normal spindles. These results suggest that additional elongation of ask1-1 spindles occurred; one possible reason for the extra-long spindles may be that it is a consequence of chromosome non-separation. We also found that normal and ask1-1 spindle lengths are clustered at discrete values, and their differences are of multiples of 0.7 microm. A search of the literature revealed that in each of several organisms, spindle lengths also differ by multiples of 0.7 microm. These findings strongly suggest that the spindle elongates in response to status of chromosome separation, and perhaps there are conserved mechanisms controlling the extent of spindle elongation.

P38αMAPK (p38α) is usually activated in response to various stresses and plays a role in the inhibition of cell proliferation and tumor progression, but little is known about its roles in meiotic spindle assembly. In this study, we characterized the dynamic localization of p38α and explored its function in mouse oocyte meiotic maturation. P38α specifically colocalized with γ-tubulin and Plk1 at the center of MTOCs and spindle poles. Depletion of p38α by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2 dephosphorylation. Notably, depletion of p38α led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased γ-tubulin and Plk1 at MTOCs. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation, and its morpholino injection almost completely rescued spindle elongation caused by p38α depletion. In addition, p38α-depletion decreased BubR1 and interfered with spindle assembly checkpoint (SAC), which resulted in aneuploid oocytes. Together, these data indicate that p38α is an important component of MTOCs, which regulates spindle assembly and spindle length, as well as stabilizes the spindle and spindle poles. Perturbed SAC and abnormal microtubule tension may be responsible for the misaligned chromosomes and high aneuploidy in p38α-depleted mouse oocytes.

Twinfilin is an evolutionarily conserved actin-binding protein, which regulates actin-dynamics in eukaryotic cells. Homologs of this protein have been detected in the genome of various protozoan parasites causing diseases in human. However, very little is known about their core functions in these organisms. We show here that a twinfilin homolog in a human pathogen Leishmania, primarily localizes to the nucleolus and, to some extent, also in the basal body region. In the dividing cells, nucleolar twinfilin redistributes to the mitotic spindle and remains there partly associated with the spindle microtubules. We further show that approximately 50% depletion of this protein significantly retards the cell growth due to sluggish progression of S phase of the cell division cycle, owing to the delayed nuclear DNA synthesis. Interestingly, overexpression of this protein results in significantly increased length of the mitotic spindle in the dividing Leishmania cells, whereas, its depletion adversely affects spindle elongation and architecture. Our results indicate that twinfilin controls on one hand, the DNA synthesis and on the other, the mitotic spindle elongation, thus contributing to karyokinesis in Leishmania.

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

Proposed origin of chimeras, mosaics and monochorionic twins

During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.

At the end of the cell cycle a cell physically divides into two daughter cells in a process called cytokinesis. Cytokinesis consists of at least four steps: 1. The position of the presumptive cytokinesis furrow is specified. 2. A contractile ring is formed. 3. The contractile ring contracts, resulting in furrow ingression. 4. Cytokinesis completes with sealing of the membranes. The mitotic spindle positions the cytokinesis furrow at the cell cortex midway along the longitudinal axis of the spindle, which is both the mid-point between the two asters and the location of the spindle midzone. The mitotic spindle emits two consecutive signals that position the furrow: Microtubule asters provide a first signal; the spindle midzone provides a second signal. Our results support the view that the spindle midzone is dispensable for completion of cytokinesis. However, the spindle midzone can negatively affect aster-positioned cytokinesis, possibly because the aster- and midzone-positioned furrows compete for contractile elements.

A key step for analysing the mechanochemistry of mitosis would be the isolation of a functional spindle capable of anaphase chromosome movement in vitro. Although Mazia and Dan first isolated spindles in 1952, with one or two possible exceptions, isolated spindles are non-functional. An alternative approach has used permeabilized cells to study anaphase chromosome movement, but these preparations are biochemically and morphologically complex, and hence difficult to analyse. We describe here a simple procedure for isolating diatom spindles which are capable of anaphase spindle elongation in vitro. With addition of ATP, the two half-spindles slide completely apart, with concomitant decrease in the zone of overlap. Electron microscopy reveals decreased numbers of microtubules throughout the spindle after ATP addition and confirms the complete absence of structures beyond the spindle poles. These results are inconsistent with theoretical models of mitosis which suggest that spindle poles are pushed apart by microtubule growth, are pulled apart by external forces applied to the poles, or are released from tension generated during spindle formation. The results are consitent with models that postulate mechanical interactions in the zone of microtubule overlap as a factor in spindle elongation.

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.

Segregation of chromosomes during cell division requires correct formation of mitotic spindles. Here, we show that a scaffold attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein-U, contributes to the attachment of spindle microtubules (MTs) to kinetochores and spindle organization. During mitosis, SAF-A was localized at the spindles, spindle midzone and cytoplasmic bridge. Depletion of SAF-A by RNA interference induced mitotic delay and defects in chromosome alignment and spindle assembly. We found that SAF-A specifically co-immunoprecipitated with the chromosome peripheral protein nucleolin and the spindle regulators Aurora-A and TPX2, indicating that SAF-A is associated with nucleolin and the Aurora-A-TPX2 complex. SAF-A was colocalized with TPX2 and Aurora-A in spindle poles and MTs. Elimination of TPX2 or Aurora-A from cells abolished the association of SAF-A with the mitotic spindle. Interestingly, SAF-A can bind to MTs and contributes to the targeting of Aurora-A to mitotic spindle MTs. Our finding indicates that SAF-A is a novel spindle regulator that plays an essential role in kinetochore-MT attachment and mitotic spindle organization.