Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.     

Tumor promoter-induced centrosome splitting in human polymorphonuclear leukocytes

Human neutrophilic polymorphonuclear leukocytes (neutrophils) adhering to a substratum undergo a dramatic change in cellular morphology when treated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). Within a few minutes of TPA treatment, the cells cease locomotion and spread symmetrically on the substratum. Concomitantly, TPA initiates centrosome splitting in a manner similar to that induced by treatment of randomly locomoting cells with a chemotactic factor. The two centrioles of a centrosome separate by a distance of several micrometers, and each of the solitary centrioles is surrounded by an aster of microtubules. Some cells also establish a third, centriole-free aster of microtubules. TPA treatment increases the total number of microtubules associated with the centrosome(s) and also increases overall polymer length. The frequency of centrosome splitting is enhanced transiently by treatment with the synthetic chemotactic peptide f-Met-Leu-Phe. Centrosome splitting is interpreted in terms of an interaction between the cell periphery and the microtubule system. Possible cellular mechanisms of this unusual phenomenon are considered.     

Microtubules' interaction with cell cortex is required for their radial organization, but not for centrosome positioning

Microtubules in interphase mammalian cells usually form a radial array with minus-ends concentrated in the central region and plus-ends placed at the periphery. This is accepted as correct, that two factors determinate the radial organization of microtubules - the centrosome, which nucleate and anchor the microtubules minus-ends, and the interaction of microtubules with cortical dynein, which positions centrosome in the cell center. However, it looks as if there are additional factors, affecting the radial structure of microtubule system. We show here that in aged Vero cytoplasts (17 h after enucleation) microtubule system lost radial organization and became chaotic. To clear up the reasons of that, we studied centrosome activity, its position in the cytoplasts and microtubule dynamics. We found that centrosome in aged cytoplasts was still active and placed in the central region of the cytoplasm, while after total disruption of the microtubules it was displaced from the center. Microtubules in aged cytoplasts were not stabilized, but they lost their ability to stop to grow near cell cortex and continued to grow reaching it. Aged cytoplast lamellae was partially depleted with dynactin though Golgi remained compact indicating dynein activity. We conclude that microtubule stoppage at cell cortex is mediated by some (protein) factors, and these factors influence radial structure of microtubule system. It seems that the key role in centrosome positioning is played by dynein complexes anchored everywhere in the cytoplasm rather than anchored in cell cortex.     

Cargo Transport by Cytoplasmic Dynein Can Center Embryonic Centrosomes

To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell’s center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.     

Evaluation of various methods of measurement of the microtubule length in the cytoplasm of cultured cells

It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 +/- 3.69 microns. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 +/- 0.72 microns long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 microns, averaging 3.33 +/- 2.43 microns; the average length of centrosomal microtubules was 1.49 +/- 0.82 microns. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of free microtubules in Vero cells actually have a length of 2-5 microns; i.e., they are much shorter than the cell radius (about 25 microns). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.     

Efficiency of organelle capture by microtubules as a function of centrosome nucleation capacity: general theory and the special case of polyspermia

Transport of organelles along microtubules is essential for the cell metabolism and morphogenesis. The presented analysis derives the probability that an organelle of a given size comes in contact with the microtubule aster. The question is asked how this measure of functionality of the microtubule aster is controlled by the centrosome. A quantitative model is developed to address this question. It is shown that for the given set of cellular parameters, such as size and total tubulin content, a centrosome nucleation capacity exists that maximizes the probability of the organelle capture. The developed general model is then applied to the capture of the female pronucleus by microtubules assembled on the sperm centrosome, following physiologically polyspermic fertilization. This application highlights an unintuitive reflection of nonlinearity of the nucleated polymerization of the cellular pool of tubulin. The prediction that the sperm centrosome should lower its nucleation capacity in the face of the competition from the other sperm is a stark illustration of the new optimality principle. Overall, the model calls attention to the capabilities of the centrosomal pathway of regulation of the transport-related functionality of the microtubule cytoskeleton. It establishes a quantitative and conceptual framework that can guide experiment design and interpretation.     

Human sperm centrosomal function during fertilization, a novel assessment for male sterility

In human fertilization, the sperm introduces the centrosome-the microtubule organizing center-and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, the sperm aster, the functioning of which is essential for pronuclear movement for the union of the male and female genomes. We established functional assay for human sperm centrosomal function, by using heterologus ICSI system with bovine and rabbit eggs. After human sperm incorporation into mammalian egg, we observed that the sperm aster was organized from sperm centrosome, and the sperm aster enlarged as the sperm nuclei underwent pronuclear formation. The normal human sperm aster formation rate at 6 h post-ICSI were 60.0% in bovine egg and 36.1% in rabbit egg, respectively. However, sperm aster formation rate following heterologus ICSI into bovine eggs with teratozoospermia (globozoospermia, dysplasia of fibrous sheath) were low. These data indicate that human sperm centrosomal function is low in abnormal shaped sperm. Wherus, elucidation of human sperm centrosomal function can lead us to find a new type of failure in "post ICSI events in fertilization".     

The dynamics of microtubule repolymerization in a cell: rapid growth from the centrosome and slow recovery of free microtubules

According to the current view, the microtubule system in animal cells consists of two components: microtubules attached to the centrosome (these microtubules stretch radially towards the cell margin), and free microtubules randomly distributed in the cytoplasm without visible association with any microtubule-organizing centers. The ratio of the two sets of microtubules in the whole microtubule array is under discussion. Addressing this question, we have analysed the recovery of microtubules in cultured Vero nucleated cells and cytoplasts, with and without centrosomes in these. Cells were fixed at different time points, and individual microtubules were traced on serial optical sections. During a slow recovery after cold treatment (4 degrees C, for 4 h; recovery at 30 degrees C) polymerization of microtubules started mainly from the centrosome. At early stages of recovery the share of free microtubules made about 10% of all microtubules, and their total length increased slower than the lenght of centrosome-attached microtubules. During a rapid recovery after nocodazole treatment (10 microg/ml, 2 h; recovery in drug-free medium at 37 degrees C), the share of free microtubules was about 35%, but their total length increased slower than the length of centrosome-attached microtubules. In 6-8 min (rapid recovery) or 12-16 min (slow recovery), tips of centrosomal microtubules reached the cell margin, and their increased density made it impossible to recognize individual microtubules. However, under the same conditions in cytoplasts without centrosomes the normal number of microtubules recovered only in 60 min, which enabled us to suppose that the complete recovery of microtubule system in the whole cells may be also rather long. When the first centrosomal microtubules reached the cell margin, the optical density of microtubules started to decrease from the centrosome region towards the cell margin, according to the exponential curve. Later on, the optical density in the centrosome region and near the cell margin remained at the same level, but microtubule density increased in the middle part of the cell, and in 45-60 min the plot of the optical density vs the distance from the centrosome became linear, as in control cells. Since no significant curling of microtubules occurs near the cell margin, the density of microtubules in the endoplasm may increase due only to polymerization of free microtubules. We suppose that in cultured cells the microtubule network recovery proceeds in two stages. At the initial stage, a rapid growth of centrosomal microtubules takes place in addition to the turnover of free microtubules with unstable minus ends. At the second stage, when microtubule growth from the centrosome becomes limited by the cell margin, a gradual extension of free microtubules occurs in the internal cytoplasm.     

Inhibition of proteasome activity impairs centrosome-dependent microtubule nucleation and organization

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.     

Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.     

Evidence for an involvement of actin in the positioning and motility of centrosomes

Cultured human polymorphonuclear leukocytes exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) spread on the substratum and undergo centrosome splitting. The two centrioles may separate by a distance of several micrometers, each being surrounded by an aster of microtubules. Here we show that the centriole/aster complexes are in constant, rapid motion through the cytoplasm, carrying with them some of the cytoplasmic granules while pushing aside others, or deforming and displacing the nucleus. An analysis of this unique motility phenomenon was undertaken. We show that intact microtubules are required for TPA-induced centrosome splitting and aster motility, but not for cell spreading. More importantly, disruption of the actin network inhibits both centrosome splitting and cell spreading, and even reverses splitting (induces convergence and fusion of asters) in polymorphonuclear leukocytes pretreated with TPA alone. These observations indicate the existence of a dynamic relationship between microtubules and actin networks and provide evidence for a role of actin in determining the position of the centrosome by way of interaction with the microtubules radiating from it.     

An Experimental and Computational Study of Effects of Microtubule Stabilization on T-Cell Polarity

T-killer cells eliminate infected and cancerous cells with precision by positioning their centrosome near the interface (immunological synapse) with the target cell. The mechanism of centrosome positioning has remained controversial, in particular the role of microtubule dynamics in it. We re-examined the issue in the experimental model of Jurkat cells presented with a T cell receptor-binding artificial substrate, which permits controlled stimulation and reproducible measurements. Neither 1-µM taxol nor 100-nM nocodazole inhibited the centrosome positioning at the “synapse” with the biomimetic substrate. At the same time, in micromolar taxol but not in nanomolar nocodazole the centrosome adopted a distinct peripheral rather than the normally central position within the synapse. This effect was reproduced in a computational energy-minimization model that assumed no microtubule dynamics, but only a taxol-induced increase in the length of the microtubules. Together, the experimental and computational results indicate that microtubule dynamics are not essential for the centrosome positioning, but that the fit of the microtubule array in the deformed body of the conjugated T cell is a major factor. The possibility of modulating the T-cell centrosome position with well-studied drugs and of predicting their effects in silico appears attractive for designing anti-cancer and antiviral therapies.     

Difference between Maturation Division and Cleavage in Starfish Oocytes: Dependency of Induced Cytokinesis on the Size of the Aster as Revealed by Transplantation of the Centrosome

Using the starfish oocyte and zygote, we investigated the abilities of the centrosome at maturation and cleavage divisions to form the aster and induce cytokinesis, in order to determine differences between these divisions. The transplanted centrosome originated from both maturation and cleavage, induced an additional furrow in cleavage in the recipient cells, but did not induce abnormal polar body formation at maturation. Although it organized an additional aster in the recipient cell in both divisions, a difference in size among asters formed was recognized. Therefore, mitotic asters were stabilized with hexylene glycol in order to measure their radius and clarify this difference. The mean radius (14.4 μm) of the first meiotic aster was significantly smaller than that (20.4 μm) of the aster at the first cleavage. The transplanted cleavage centrosome formed as small an aster as the recipient's own at maturation divisions. When zygotes were briefly treated with colcemid so that the zygotes could not perform cytokinesis but did perform karyokinesis, the size of aster became the same as that in meiosis. These results prove that although any centrosome functions as a microtubule organizing center independent of its origin, the size of the resultant aster decides whether or not cytokinesis would be induced.     

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

Protoplast isolation,development, and regeneration in different strains ofPilayella littoralis (L.) Kjellm. (Phaeophyceae)

Summary Using an antibody against tyrosinated tubulin and epifluorescence microscopy, mitosis was studied in three different microvessel endothelial cell types recently isolated from bovine corpus luteum. Dividing cells were flat and at certain stages individual microtubules could be followed for considerable lengths. The structure of the spindle apparatus and the course of mitosis were conventional. Microtubule asters were small from prophase until metaphase in all three cell types. However, whereas in two cell types telophase asters remained inconspicuous, prominent asters, of mostly straight microtubules, formed in telophase cells of a third cell type. Thus, aster size is heterogeneous between different endothelial cell types. Large microtubule asters are not regularly found in dividing cultured mammalian cells. The microendothelial cell types present themselves as appropriate systems for spindle research and especially for the study of aster microtubule dynamics and function.     

Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis

Katanin is a heterodimer that exhibits ATP-dependent microtubule-severing activity in vitro. In Xenopus egg extracts, katanin activity correlates with the addition of cyclin B/cdc2, suggesting a role for microtubule severing in the disassembly of long interphase microtubules as the cell prepares for mitosis. However, studies from plant cells, cultured neurons, and nematode embryos suggest that katanin could be required for the organization or postnucleation processing of microtubules, rather than the dissolution of microtubule structures. Here we reexamine katanin's role by studying acentrosomal female meiotic spindles in C. elegans embryos. In mutant embryos lacking katanin, microtubules form around meiotic chromatin but do not organize into bipolar spindles. By using electron tomography, we found that katanin converts long microtubule polymers into shorter microtubule fragments near meiotic chromatin. We further show that turning on katanin during mitosis also creates a large pool of short microtubules near the centrosome. Furthermore, the identification of katanin-dependent microtubule lattice defects supports a mechanism involving an initial perforation of the protofilament wall. Taken together, our data suggest that katanin is used during meiotic spindle assembly to increase polymer number from a relatively inefficient chromatin-based microtubule nucleation pathway.     

Centrosome reorientation in wound-edge cells is cell type specific

The reorientation of the microtubule organizing center during cell migration into a wound in the monolayer was directly observed in living wound-edge cells expressing gamma-tubulin tagged with green fluorescent protein. Our results demonstrate that in CHO cells, the centrosome reorients to a position in front of the nucleus, toward the wound edge, whereas in PtK cells, the centrosome lags behind the nucleus during migration into the wound. In CHO cells, the average rate of centrosome motion was faster than that of the nucleus; the converse was true in PtK cells. In both cell lines, centrosome motion was stochastic, with periods of rapid motion interspersed with periods of slower motion. Centrosome reorientation in CHO cells required dynamic microtubules and cytoplasmic dynein/dynactin activity and could be prevented by altering cell-to-cell or cell-to-substrate adhesion. Microtubule marking experiments using photoactivation of caged tubulin demonstrate that microtubules are transported in the direction of cell motility in both cell lines but that in PtK cells, microtubules move individually, whereas their movement is more coherent in CHO cells. Our data demonstrate that centrosome reorientation is not required for directed migration and that diverse cells use distinct mechanisms for remodeling the microtubule array during directed migration.     

Microtubules in ascidian eggs during meiosis, fertilization, and mitosis

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.     

Identification of Microtubule-Organizing Centers in Interphase Melanophores of Xenopus Laevis Larvae In Vivo

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immuno-stained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle.

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.