Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. We show that this "internal" matrix is a distinct structure from the microtubule spindle and from a lamin B-containing spindle envelope. By injection of 2000-kDa dextran, we show that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as "struts" stretching the spindle matrix. In addition, we demonstrate that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division.     

CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.     

Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

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

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

Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake

During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)-dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.     

The formation and functioning of yeast mitotic spindles

The mitotic spindle contains the machinery responsible for sister chromatid segregation. It is composed of a complex and dynamic array of microtubules, which are nucleated from the spindle poles. Studies of yeast spindle functions by molecular genetic analysis and by in vitro functional analysis have identified proteins that are mitosis-specific and present at very low concentrations in the cell, and have revealed the molecular bases of several processes required for the formation and functioning of the mitotic spindle. Here I review the current knowledge of the processes that are common to most eukaryotes: microtubule nucleation at the spindle poles, bipolar spindle assembly, maintenance of the spindle structure, chromosome attachment to the spindle and chromosome separation on the spindle.     

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

Transient defects of mitotic spindle geometry and chromosome segregation errors

ABSTRACT: Assembly of a bipolar mitotic spindle is essential to ensure accurate chromosome segregation and prevent aneuploidy, and severe mitotic spindle defects are typically associated with cell death. Recent studies have shown that mitotic spindles with initial geometric defects can undergo specific rearrangements so the cell can complete mitosis with a bipolar spindle and undergo bipolar chromosome segregation, thus preventing the risk of cell death associated with abnormal spindle structure. Although this may appear as an advantageous strategy, transient defects in spindle geometry may be even more threatening to a cell population or organism than permanent spindle defects. Indeed, transient spindle geometry defects cause high rates of chromosome mis-segregation and aneuploidy. In this review, we summarize our current knowledge on two specific types of transient spindle geometry defects (transient multipolarity and incomplete spindle low separation) and describe how these mechanisms cause chromosome mis-segregation and aneuploidy. Finally, we discuss how these transient spindle defects may specifically contribute to the chromosomal instability observed in cancer cells.     

Skeletor, a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix

A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor antibody labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.     

Stable kinetochore–microtubule attachments restrict MTOC position and spindle elongation in oocytes

In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball‐like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore–microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore–microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore–microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles. These findings suggest a role for stable kinetochore–microtubule attachments in fine‐tuning acentrosomal spindle bipolarity.     

Control of spindle form and function in grasshopper spermatocytes

The influence of the mitotic organizing centers, the kinetochores and the polar organizers, in controlling the dynamic spindle form and function has been investigated in the primary spermatocytes of two grasshoppers, Arphia xanthoptera and Melanoplus differentialis. A new measure of the total birefringent material in the spindle is introduced—volume-birefringence. This measure avoids many of the problems associated with the traditional retardation measurements of spindle organization.—The number of chromosomes (and their kinetochores) in a spindle can be altered with a piezoelectric micromanipulator in three ways: 1) chromosomes can be removed permanently from the cell, 2) chromosomes can be detached from the spindle and allowed to reenter the spindle at a later time, and 3) chromosomes can be transferred from one spindle to another in cells containing two spindles. Such operations show the volume-birefringence of the spindle is proportional to the number of chromosomes in the spindle. A residual volume-birefringence is seen and attributed to the contribution of the polar organizers to spindle structure. The relative polar contribution differs in the two species. Chromosome motion and spindle elongation in anaphase are unaffected by the number of chromosomes in the spindle. The proportion of volume-birefringence associated with a kinetochore is used to estimate the number of microtubules one might expect to see if the birefringence of the spindle is of microtubular origin. These calculations predict about twice the number of microtubules per kinetochore than seen with the electron microscope. Reasons are suggested to explain this discrepancy.— It is argued that chromosome detachment releases spindle component subunits into the total subunit pool, but that these excess subunits do not influence the metaphase form nor the anaphase function of the spindle; therefore, spindle dynamics are under the direct control of the kinetochores and the polar organizing centers.     

Directly probing the mechanical properties of the spindle and its matrix

Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.     

The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.     

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell. It is unknown whether spindle alignment requires both apical and basal cues, nor have molecular motors been identified that regulate spindle movement. Using live imaging of neuroblasts within intact larval brains, we detect independent movement of both apical and basal spindle poles, suggesting that forces act on both poles. We show that reducing astral microtubules decreases the frequency of spindle movement, but not its maximum velocity, suggesting that one or few microtubules can move the spindle. Mutants in the Lis1/dynactin complex strongly decrease maximum and average spindle velocity, consistent with this motor complex mediating spindle/cortex forces. Loss of either astral microtubules or Lis1/dynactin leads to spindle/cortical polarity alignment defects at metaphase, but these are rescued by telophase. We propose that an early Lis1/dynactin-dependent pathway and a late Lis1/dynactin-independent pathway regulate neuroblast spindle orientation.     

Micromechanics of the Vertebrate Meiotic Spindle Examined by Stretching along the Pole-to-Pole Axis

The meiotic spindle is a bipolar molecular machine that is designed to segregate duplicated chromosomes toward the opposite poles of the cell. The size and shape of the spindle are considered to be maintained by a balance of forces produced by molecular motors and microtubule assembly dynamics. Several studies have probed how mechanical perturbations of the force balance affect the spindle structure. However, the spindle’s response to a stretching force acting at the spindle pole and along its long axis, i.e., the direction in which chromosomes are segregated, has not been examined. Here, we describe a method to apply a stretching force to the metaphase spindle assembled in Xenopus egg extracts and measure the relationship between the force and the three-dimensional deformation of the spindle. We found that the spindle behaves as a Zener-type viscoelastic body when forces are applied at the spindle pole, generating a restoring force for several minutes. In addition, both the volume of the spindle and the tubulin density are conserved under the stretching force. These results provide insight into how the spindle size is maintained at metaphase.     

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.     

Micromanipulation studies of the mitotic apparatus in sand dollar eggs

Mechanical properties of the mitotic spindle and the effects of various operations of the mitotic apparatus on the chromosome movement and spindle elongation were investigated in fertilized eggs and blastomeres of the sand dollar, Clypeaster japonicus. On the basis of results with mechanical stretching and compression of the spindle with a pair of microneedles and the behavior of an oil drop microinjected into the spindle, it was concluded that the equatorial region of the spindle is mechanically weaker than the half-spindle region. Anaphase chromosome movement occurred in the spindle from which an aster had been removed or separated with its polar end and in the spindle in which the interzonal region had been removed. This fact indicates that chromosomes move poleward in anaphase by forces generated near the kinetochores in the half-spindle. Because of the effects of separation or removal of an aster from the spindle on the spindle elongation in anaphase and the behavior of the aster, it was concluded that the spindle elongation in anaphase is caused by pulling forces generated by asters attached to the ends of the spindle.     

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.