Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body

The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72-Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.     

Requirement of Aurora-A kinase in astral microtubule polymerization and spindle microtubule flux

Mitotic Aurora-A kinase was found to be required for formation of bipolar spindle, ensuring accurate chromosome segregation in mitosis. Recently, Aurora-A was shown to promote Ran-GTP-induced spindle formation and astral microtubule development. Here, by selective immunodepletion, we showed that Aurora-A was required for centrosome- but not Ran-GTP-induced astral microtubule formation in Xenopus egg extracts. Aurora-A enhanced microtubule polymerization in both centrosome- and Ran-GTP-induced aster assemblies: shortening the timing of aster assembly and increasing the aster size. Indeed, adding of Aurora-A protein alone induced microtubule clustering, which was abrogated by Aurora kinase inhibitory small molecule ZM447439. In addition, we showed that Aurora-A was indispensable for Ran-GTP-induced bipolar spindle formation. Inhibition of Aurora-A activity by adding of kinase inactive dominant mutant led to spindle collapse and formation of monopolar spindle whereas minus-end motor protein dynein/dynactin inhibitor p50/dynamitin rescued the bipolar structure. Lastly, we revealed that Aurora-A was necessary for microtubule poleward flux and this requirement depended on kinase activity. Thus, we showed that Aurora-A promoted microtubule polymerization and maintained microtubule flux in ensuring proper bipolar spindle assembly.     

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore–microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.     

Reorientation of mispositioned spindles in short astral microtubule mutant spc72Delta is dependent on spindle pole body outer plaque and Kar3 motor protein

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic gamma-tubulin complex, can only generate very short (<1 microm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.     

Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.     

Components of the yeast spindle and spindle pole body

Yeast spindle pole bodies (SPBs) with attached nuclear microtubles were enriched approximately 600-fold from yeast cell extracts. 14 mAbs prepared against this enriched SPB fraction define at least three components of the SPB and spindle. Immunofluorescent staining of yeast cells showed that throughout the cell cycle two of the components (110 and 90 kD) were localized exclusively to the SPB region, and the other (80 kD) was localized both to the SPB region and to particulate dots in short spindles. Immunoelectron microscopy confirmed and extended most of these findings. Thus the 110-kD component was localized to a layer in the SPB just to the nuclear side of the plane of the inner nuclear membrane. The 90-kD component was localized in a layer across the cytoplasmic face of intact SPBs, and, in SPBs where nuclear microtubules were removed by extraction with DEAE-dextran, the 90-kD component was also found in an inner nuclear layer close to where spindle microtubules emerge. In intact SPBs with attached nuclear microtubules the anit-80-kD mAb labels microtubules, particularly those close to the SPB. These results begin to provide a preliminary molecular map of the SPB and should also enable the corresponding genes to be isolated.     

Regional variation of microtubule flux reveals microtubule organization in the metaphase meiotic spindle

Continuous poleward movement of tubulin is a hallmark of metaphase spindle dynamics in higher eukaryotic cells and is essential for stable spindle architecture and reliable chromosome segregation. We use quantitative fluorescent speckle microscopy to map with high resolution the spatial organization of microtubule flux in Xenopus laevis egg extract meiotic spindles. We find that the flux velocity decreases near spindle poles by ~20%. The regional variation is independent of functional kinetochores and centrosomes and is suppressed by inhibition of dynein/dynactin, kinesin-5, or both. Statistical analysis reveals that tubulin flows in two distinct velocity modes. We propose an association of these modes with two architecturally distinct yet spatially overlapping and dynamically cross-linked arrays of microtubules: focused polar microtubule arrays of a uniform polarity and slower flux velocities are interconnected by a dense barrel-like microtubule array of antiparallel polarities and faster flux velocities.     

The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage

The yeast protein Stu2 belongs to the XMAP215 family of conserved microtubule-binding proteins which regulate microtubule plus end dynamics. XMAP215-related proteins also bind to centrosomes and spindle pole bodies (SPBs) through proteins like the mammalian transforming acidic coiled coil protein TACC or the yeast Spc72. We show that yeast Spc72 has two distinct domains involved in microtubule organization. The essential 100 N-terminal amino acids of Spc72 interact directly with the gamma-tubulin complex, and an adjacent non-essential domain of Spc72 mediates binding to Stu2. Through these domains, Spc72 brings Stu2 and the gamma-tubulin complex together into a single complex. Manipulation of Spc72-Stu2 interaction at SPBs compromises the anchorage of astral microtubules at the SPB and surprisingly also influences the dynamics of microtubule plus ends. Permanently tethering Stu2 to SPBs by fusing it to a version of Spc72 that lacks the Stu2-binding site in part complements these defects in a manner which is dependent upon the microtubule-binding domain of Stu2. Thus, the SPB-associated Spc72-Stu2 complex plays a key role in regulating microtubule properties.     

Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.     

Dynamics of the spindle pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-substitution electron microscopy

Cryptococcus neoformans is an opportunistic human pathogen belonging to basidiomycetous fungi and has unique properties in cell cycle progression. In the present study, dynamics of the spindle pole body (SPB) during the cell cycle was examined using freeze-substitution and serial thin-sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar-shaped in G1 through G2 phases. At the beginning of prophase, globular elements of the SPB enlarged, associated with numerous cytoplasmic microtubules, and separated on the nuclear envelope. At prometaphase, the SPBs entered the nuclear region by breaking a part of the nuclear membrane, were located at the isthmus, and were associated with numerous nuclear microtubules. The nuclear division process was carried out in the daughter cell, though the nucleolus remained in the mother cell. At anaphase, one half of the nucleus returned to the mother cell. At telophase, the SPB element was extruded back to the cytoplasm from the nuclear region. By analyzing serial sections of 63 cells, duplication of the SPB was found to take place in the early G1 phase. Thus, the location, structure, and duplication cycle of the C. neoformans SPB are different from those of Saccharomyces cerevisiae , but have similarities to those of Schizosaccharomyces pombe .     

Asymmetric mitotic segregation of the yeast spindle pole body.

The yeast KAR1 gene is required for spindle pole body (SPB) duplication and nuclear fusion. We determine here that KAR1-beta-galactosidase hybrid proteins localize to the outer face of the SPB. Remarkably, after SPB duplication, the hybrid protein was found associated with only one of the two SPBs, usually the one that enters the bud. Using an ndc1 mutant, which forms a defective SPB at the nonpermissive temperature, we found that the hybrid was exclusively associated with the "new" SPB. Two regions of KAR1 contribute to its localization; an internal 70 residue region was necessary and sufficient to localize hybrids to the SPB, and the hydrophobic carboxyl terminus localized proteins to the nuclear envelope. The localization domains correspond to two functional domains required for SPB duplication. We suggest that KAR1 is anchored to the nuclear envelope and interacts with at least one other SPB component during the cell cycle.     

Microtubule organization by the budding yeast spindle pole body.

In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.     

Lessons from yeast: the spindle pole body and the centrosome

The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly established. This involved a large variety of techniques which are described here, and the potential use of some of these in the centrosome field is highlighted. In particular, very useful structural information on the SPB was obtained from a reconstituted complex, the γ-tubulin complex, and also from a sub-particle, SPB cores, prepared by extraction of an enriched SPB preparation. The labelling of SPB proteins with GFP at the N or C termini, using GFP tags inserted into the genome, gave informative electron microscopy localization and fluorescence resonance energy transfer data. Examples are given of more precise functional data obtained by removing domains from one SPB protein, Spc110p, without affecting its essential function. Finally, a structural model for SPB duplication is described and the differences between SPB and centrosome duplication discussed.     

A new method reveals microtubule minus ends throughout the meiotic spindle

Anastral meiotic spindles are thought to be organized differently from astral mitotic spindles, but the field lacks the basic structural information required to describe and model them, including the location of microtubule-nucleating sites and minus ends. We measured the distributions of oriented microtubules in metaphase anastral spindles in Xenopus laevis extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We found that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. Based on these data, we propose a model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles.     

XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly

BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.     

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.     

Mechanisms of genetic instability revealed by analysis of yeast spindle pole body duplication.

Aneuploidy and polyploidy are commonly observed in transformed cells. These states arise from failures during mitotic chromosome segregation, some of which can be traced to defects in the function or duplication of the centrosome. The centrosome is the organizing center for the mitotic spindle, and the equivalent organelle in the budding yeast, Saccharomyces cerevisiae, is the spindle pole body. We review how defects in spindle pole body duplication or function lead to genetic instability in yeast. There are several well documented instances of genetic instability in yeast that can be traced to the spindle pole body, all of which serve as models for genetic instability in transformed cells.     

Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.