Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.     

Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein–dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.     

Mechanisms of spindle-pole organization are influenced by kinetochore activity in mammalian cells

The spindle is a fusiform bipolar-microtubule array that is responsible for chromosome segregation during mitosis. Focused poles are an essential feature of spindles in vertebrate somatic cells, and pole focusing has been shown to occur through a centrosome-independent self-organization mechanism where microtubule motors cross-link and focus microtubule minus ends. Most of our understanding of this mechanism for pole focusing derives from studies performed in cell-free extracts devoid of centrosomes and kinetochores. Here, we examine how sustained force from kinetochores influences the mechanism of pole focusing in cultured cells. We show that the motor-driven self-organization activities associated with NuMA (i.e., cytoplasmic dynein) and HSET are not necessary for pole focusing if sustained force from kinetochores is inhibited in Nuf2- or Mis12-deficient cells. Instead, pole organization relies on TPX2 as it cross-links spindle microtubules to centrosome-associated mitotic asters. Thus, both motor-driven and static-cross-linking mechanisms contribute to spindle-pole organization, and kinetochore activity influences the mechanism of spindle-pole organization. The motor-driven self-organization of microtubule minus ends at spindle poles is needed to organize spindle poles in vertebrate somatic cells when kinetochores actively exert force on spindle microtubules.     

Spatial regulation improves antiparallel microtubule overlap during mitotic spindle assembly

The mitotic spindle plays an essential role in chromosome segregation during cell division. Spindle formation and proper function require that microtubules with opposite polarity overlap and interact. Previous computational simulations have demonstrated that these antiparallel interactions could be created by complexes combining plus- and minus-end-directed motors. The resulting spindles, however, exhibit sparse antiparallel microtubule overlap with motor complexes linking only a nominal number of antiparallel microtubules. Here we investigate the role that spatial differences in the regulation of microtubule interactions can have on spindle morphology. We show that the spatial regulation of microtubule catastrophe parameters can lead to significantly better spindle morphology and spindles with greater antiparallel MT overlap. We also demonstrate that antiparallel microtubule overlap can be increased by having new microtubules nucleated along the length of existing astral microtubules, but this increase negatively affects spindle morphology. Finally, we show that limiting the diffusion of motor complexes within the spindle region increases antiparallel microtubule interaction.     

Arabidopsis thaliana protein,ATK1, is a minus-end directed kinesin that exhibits non-processive movement

The microtubule cytoskeleton forms the scaffolding of the meiotic spindle. Kinesins, which bind to microtubules and generate force via ATP hydrolysis, are also thought to play a critical role in spindle assembly, maintenance, and function. The A. thaliana protein, ATK1 (formerly known as KATA), is a member of the kinesin family based on sequence similarity and is implicated in spindle assembly and/or maintenance. Thus, we want to determine if ATK1 behaves as a kinesin in vitro, and if so, determine the directionality of the motor activity and processivity character (the relationship between molecular "steps" and microtubule association). The results show that ATK1 supports microtubule movement in an ATP-dependent manner and has a minus-end directed polarity. Furthermore, ATK1 exhibits non-processive movement along the microtubule and likely requires at least four ATK1 motors bound to the microtubule to support movement. Based on these results and previous data, we conclude that ATK1 is a non-processive, minus-end directed kinesin that likely plays a role in generating forces in the spindle during meiosis.     

Still entangled: Assembly of the central spindle by multiple microtubule modulators

The central spindle is a microtubule-based structure that assembles during anaphase of mitosis in animal cells and is essential for multiple steps of cytokinesis. Central spindle assembly occurs by the cooperative action of multiple microtubule motors and modulators. Here, we review the mechanism by which the central spindle is formed, the role of several key proteins in this process and how central spindle assembly is temporally and spatially coordinated with mitosis.     

A Force Balance Model of Early Spindle Pole Separation in Drosophila Embryos

The formation and function of the mitotic spindle depends upon force generation by multiple molecular motors and by the dynamics of microtubules, but how these force-generating mechanisms relate to one another is unclear. To address this issue we have modeled the separation of spindle poles as a function of time during the early stages of spindle morphogenesis in Drosophila embryos. We propose that the outward forces that drive the separation of the spindle poles depend upon forces exerted by cortical dynein and by microtubule polymerization, and that these forces are antagonized by a C-terminal kinesin, Ncd, which generates an inward force on the poles. We computed the sum of the forces generated by dynein, microtubule polymerization, and Ncd, as a function of the extent of spindle pole separation and solved an equation relating the rate of pole separation to the net force. As a result, we obtained graphs of the time course of spindle pole separation during interphase and prophase that display a reasonable fit to the experimental data for wild-type and motor-inhibited embryos. Among the novel contributions of the model are an explanation of pole separation after simultaneous loss of Ncd and dynein function, and the prediction of a large value for the effective centrosomal drag that is needed to fit the experimental data. The results demonstrate the utility of force balance models for explaining certain mitotic movements because they explain semiquantitatively how the force generators drive a rapid initial burst of pole separation when the net force is great, how pole separation slows down as the force decreases, and how a stable separation of the spindle poles characteristic of the prophase steady state is achieved when the force reaches zero.     

Pericentric chromatin is an elastic component of the mitotic spindle

BACKGROUND: Prior to chromosome segregation, the mitotic spindle bi-orients and aligns sister chromatids along the metaphase plate. During metaphase, spindle length remains constant, which suggests that spindle forces (inward and outward) are balanced. The contribution of microtubule motors, regulators of microtubule dynamics, and cohesin to spindle stability has been previously studied. In this study, we examine the contribution of chromatin structure on kinetochore positioning and spindle-length control. After nucleosome depletion, by either histone H3 or H4 repression, spindle organization was examined by live-cell fluorescence microscopy. RESULTS: Histone repression led to a 2-fold increase in sister-centromere separation and an equal increase in metaphase spindle length. Histone H3 repression does not impair kinetochores, whereas H4 repression disrupts proper kinetochore function. Deletion of outward force generators, kinesins Cin8p and Kip1p, shortens the long spindles observed in histone-repressed cells. Oscillatory movements of individual sister chromatid pairs are not altered after histone repression. CONCLUSIONS: The increase in spindle length upon histone repression and restoration of wild-type spindle length by the loss of plus-end-directed motors suggests that during metaphase, centromere separation and spindle length are governed in part by the stretching of pericentric chromatin. Chromatin is an elastic molecule that is stretched in direct opposition to the outward force generators Cin8p and Kip1p. Thus, we assign a new role to chromatin packaging as an integral biophysical component of the mitotic apparatus.     

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity—a mechanical paradigm that may be important throughout the spindle.     

Mitotic functions of kinesin-5

In all eukaryotic cells, molecular motor proteins play essential roles in spindle assembly and function. The homotetrameric kinesin-5 motors in particular generate outward forces that establish and maintain spindle bipolarity and contribute to microtubule flux. Cell-cycle dependent phosphorylation of kinesin-5 motors regulates their localization to the mitotic spindle. Analysis of live cells further shows that kinesin-5 motors are highly dynamic in the spindle. Understanding the interactions of kinesin-5 motors with microtubules and other spindle proteins is likely to broaden the documented roles of kinesin-5 motors during cell division.     

Chromosome movement in mitosis requires microtubule anchorage at spindle poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly

During cytokinesis, the organization of the spindle midzone and chromosome segregation is controlled by the central spindle, a microtubule cytoskeleton containing kinesin motors and non‐motor microtubule‐associated proteins. The anaphase spindle elongation 1/protein regulator of cytokinesis 1/microtubule associated protein 65 (Ase1/PRC1/MAP65) family of microtubule‐bundling proteins are key regulators of central spindle assembly, mediating microtubule crosslinking and spindle elongation in the midzone. Ase1/PRC1/MAP65 serves as a complex regulatory platform for the recruitment of other midzone proteins at the spindle midzone. Herein, we summarize recent advances in understanding of the structural domains and molecular kinetics of the Ase1/PRC1/MAP65 family. We summarize the regulatory network involved in post‐translational modifications of Ase1/PRC1 by cyclin‐dependent kinase 1 (Cdk1), cell division cycle 14 (Cdc14) and Polo‐like kinase 1 (Plk1) and also highlight multiple functions of Ase1/PRC1 in central spindle organization, spindle elongation and cytokinesis during cell division.     

Roles of two homotetrameric kinesins in sea urchin embryonic cell division

To improve our understanding of the roles of microtubule cross-linking motors in mitosis, we analyzed two sea urchin embryonic kinesin-related proteins. It is striking to note that both of these proteins behave as homotetramers, but one behaves as a more compact molecule than the other. These observations suggest that these two presumptive motors could cross-link microtubules into bundles with different spacing. Both motors localize to mitotic spindles, and antibody microinjection experiments suggest that they have mitotic functions. Thus, one of these kinesin-related proteins may cross-link spindle microtubules into loose bundles that are "tightened" by the other.     

NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.     

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.     

Chromokinesins: localization-dependent functions and regulation during cell division

The bipolar spindle is a highly dynamic structure that assembles transiently around the chromosomes and provides the mechanical support and the forces required for chromosome segregation. Spindle assembly and chromosome movements rely on the regulation of microtubule dynamics and a fine balance of forces exerted by various molecular motors. Chromosomes are themselves central players in spindle assembly. They generate a RanGTP gradient that triggers microtubule nucleation and stabilization locally and they interact dynamically with the microtubules through motors targeted to the chromatin. We have previously identified and characterized two of these so-called chromokinesins: Xkid (kinesin 10) and Xklp1 (kinesin 4). More recently, we found that Hklp2/kif15 (kinesin 12) is targeted to the chromosomes through an interaction with Ki-67 in human cells and is therefore a novel chromokinesin. Hklp2 also associates with the microtubules specifically during mitosis, in a TPX2 (targeting protein for Xklp2)-dependent manner. We have shown that Hklp2 participates in spindle pole separation and in the maintenance of spindle bipolarity in metaphase. To better understand the function of Hklp2, we have performed a detailed domain analysis. Interestingly, from its positioning on the chromosome arms, Hklp2 seems to restrict spindle pole separation. In the present review, we summarize the current knowledge of the function and regulation of the different kinesins associated with chromosome arms during cell division, including Hklp2 as a novel member of this so-called chromokinesin family.     

And the dead shall rise: actin and myosin return to the spindle

The spindle directs chromosome partitioning in eukaryotes and, for the last three decades, has been considered primarily a structure based on microtubules, microtubule motors, and other microtubule binding proteins. However, a surprisingly large body of both old and new studies suggests roles for actin filaments (F-actin) and myosins (F-actin-based motor proteins) in spindle assembly and function. Here we review these data and conclude that in several cases the evidence for the participation of F-actin and myosins in spindle function is very strong, and in the situations where it is less strong, there is nevertheless enough evidence to warrant further investigation.     

Developmental regulation of central spindle assembly and cytokinesis during vertebrate embryogenesis

Mitosis and cytokinesis not only ensure the proper segregation of genetic information but also contribute importantly to morphogenesis in embryos. Cytokinesis is controlled by the central spindle, a microtubule-based structure containing numerous microtubule motors and microtubule-binding proteins, including PRC1. We show here that central spindle assembly and function differ dramatically between two related populations of epithelial cells in developing vertebrate embryos examined in vivo. Compared to epidermal cells, early neural epithelial cells undergo exaggerated anaphase chromosome separation, rapid furrowing, and a marked reduction of microtubule density in the spindle midzone. Cytokinesis in normal early neural epithelial cells thus resembles that in cultured vertebrate cells experimentally depleted of PRC1. We find that PRC1 mRNA and protein expression is surprisingly dynamic in early vertebrate embryos and that neural-plate cells contain less PRC1 than do epidermal cells. Expression of excess PRC1 ameliorates both the exaggerated anaphase and reduced midzone microtubule density observed in early neural epithelial cells. These PRC1-mediated modifications to the cytokinetic mechanism may be related to the specialization of the midbody in neural cells. These data suggest that PRC1 is a dose-dependent regulator of the central spindle in vertebrate embryos and demonstrate unexpected plasticity to fundamental mechanisms of cell division.     

Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.