Interphase microtubules determine the initial alignment of the mitotic spindle

In the fission yeast Schizosaccharomyces pombe, interphase microtubules (MTs) position the nucleus [1, 2], which in turn positions the cell-division plane [1, 3]. It is unclear how the spindle orients, with respect to the predetermined division plane, to ensure that the chromosomes are segregated across this plane. It has been proposed that, during prometaphase, the astral MT interaction with the cell cortex aligns the spindle with the cell axis [4] and also participates in a spindle orientation checkpoint (SOC), which delays entry into anaphase as long as the spindle is misaligned [5-7]. Here, we trace the position of the spindle throughout mitosis in a single-cell assay. We find no evidence for the SOC. We show that the spindle is remarkably well aligned with the cell longitudinal axis at the onset of mitosis, by growing along the axis of the adjacent interphase MT. Misalignment of nascent spindles can give rise to anucleate cells when spindle elongation is impaired. We propose a new role for interphase microtubules: through interaction with the spindle pole body, interphase microtubules determine the initial alignment of the spindle in the subsequent cell division.     

Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3-dependent checkpoint in fission yeast

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.     

A Highlights from MBoC Selection: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell''s height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.     

Intra-nuclear microtubules and a mitotic spindle orientation checkpoint

Cells of the fission yeast Schizosaccharomyces pombe have a checkpoint mechanism that reportedly monitors the orientation of the mitotic spindle. Astral microtubules in pre-anaphase spindles are thought to contact the contractile actin ring at the plasma membrane in order to rotate the spindle and to sense spindle orientation. Here, we show that these microtubules are actually inside the nuclear envelope.     

Mechanism controlling perpendicular alignment of the spindle to the axis of cell division in fission yeast

In animal cells, the mitotic spindle is aligned perpendicular to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring (CAR). We show that, in fission yeast, spindle rotation is dependent on the interaction of astral microtubules with the cortical actin cytoskeleton. Interaction initially occurs with a region surrounding the nucleus, which we term the astral microtubule interaction zone (AMIZ). Simultaneous contact of astral microtubules from both poles with the AMIZ directs spindle rotation and this requires both actin and two type V myosins, Myo51 and Myo52. Astral microtubules from one pole only then contact the CAR, which is located at the centre of the AMIZ. We demonstrate that the anillin homologue Mid1, which dictates correct placement of the CAR, is necessary to stabilise the mitotic spindle perpendicular to the axis of cell division. Finally, we show that the position of the mitotic spindle is monitored by a checkpoint that regulates the timing of sister chromatid separation.     

The roles of fission yeast ase1 in mitotic cell division, meiotic nuclear oscillation, and cytokinesis checkpoint signaling

The Ase1/Prc1 proteins constitute a conserved microtubule-associated protein family that is implicated in central spindle formation and cytokinesis. Here we characterize a role for fission yeast Ase1. Ase1 localizes to microtubule overlapping zones and displays dynamic alterations of localization during the cell cycle. In particular, its spindle localization during metaphase is reduced substantially, followed by robust appearance at the spindle midzone in anaphase. ase1 deletions are viable but defective in nuclear and septum positioning and completion of cytokinesis, which leads to diploidization and chromosome loss. Time-lapse imaging shows that elongating spindles collapse abruptly in the middle of anaphase B. Either absence or overproduction of Ase1 results in profound defects on microtubule bundling in an opposed manner, indicating that Ase1 is a dose-dependent microtubule-bundling factor. In contrast microtubule nucleating activities are not noticeably compromised in ase1 mutants. During meiosis astral microtubules are not bundled and oscillatory nuclear movement is impaired significantly. The Aurora kinase does not correctly localize to central spindles in the absence of Ase1. Finally Ase1 acts as a regulatory component in the cytokinesis checkpoint that operates to inhibit nuclear division when the cytokinesis apparatus is perturbed. Ase1, therefore, couples anaphase completion with cytokinesis upon cell division.     

Positioning and elongation of the fission yeast spindle by microtubule-based pushing

In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.     

Monastral bipolar spindles in meiosis II of male Trichosia pubescens (Sciaridae): early stages of spindle formation and chromosome orientation.

The metaphase spindle of male meiosis II in fungus gnats (Sciaridae) is one example of naturally occurring monastral bipolar spindles. To gain further insights into how the bipolar spindle is formed in the presence of only one polar center, prometaphase of male meiosis II was investigated in the sciarid Trichosia pubescens by means of anti-tubulin immunofluorescence, DAPI chromosome staining, and electron microscopy of ultrathin serial sections. The first step in spindle formation after interkinesis seems to be the organization of an astral half-spindle, probably by MTOC activity of the astral region. With the exception of the non-disjunctional X chromosome, which always lies close to the aster, the chromosomes are found to occupy various positions with respect to the astral region, revealing different orientations of their chromatid kinetochores. It was observed that some of the mal-oriented kinetochores are associated with microtubules that, due to their orientation perpendicular to the spindle axis, are unlikely to originate from the astral region. Therefore, these mal-oriented microtubules are taken as an indication of a dispersed MTOC activity near the chromosomes or at kinetochores. According to recent models of chromosome-induced spindle self-organization [e.g., Merdes et al., 1997: J. Cell Biol. 138:953-956], they could be responsible for the formation of the other (anastral) half-spindle and for amphitelic (bipolar) orientation of the chromosomes.     

TINA interacts with the NIMA kinase in Aspergillus nidulans and negatively regulates astral microtubules during metaphase arrest

The tinA gene of Aspergillus nidulans encodes a protein that interacts with the NIMA mitotic protein kinase in a cell cycle-specific manner. Highly similar proteins are encoded in Neurospora crassa and Aspergillus fumigatus. TINA and NIMA preferentially interact in interphase and larger forms of TINA are generated during mitosis. Localization studies indicate that TINA is specifically localized to the spindle pole bodies only during mitosis in a microtubule-dependent manner. Deletion of tinA alone is not lethal but displays synthetic lethality in combination with the anaphase-promoting complex/cyclosome mutation bimE7. At the bimE7 metaphase arrest point, lack of TINA enhanced the nucleation of bundles of cytoplasmic microtubules from the spindle pole bodies. These microtubules interacted to form spindles joined in series via astral microtubules as revealed by live cell imaging. Because TINA is modified and localizes to the spindle pole bodies at mitosis, and lack of TINA causes enhanced production of cytoplasmic microtubules at metaphase arrest, we suggest TINA is involved in negative regulation of the astral microtubule organizing capacity of the spindle pole bodies during metaphase.     

Spindle positioning in fibroblasts supports an astral microtubule length dependent force generation at the basal membrane.

V79 Chinese hamster fibroblasts that maintain an elongated shape in metaphase occur at a low frequency and often show the spindle asymmetrically positioned. We show here that this aberrant position is corrected in anaphase by an external force, pulling the spindle into place. The force was applied on astral microtubules because spindle motility was hampered when astral microtubules were poorly developed spontaneously, or destroyed by colcemid. Colcemid also abolished the observed downward positioning of centrosomes in anaphase. One pole of the spindle was usually dominant during correction, but occasionally both poles could become subject to pulling making the spindle move perpendicular to the long axis of the cell, which induced reshaping of the cell. The pulling force acted unevenly with short intervals of resting between the pulling. Spindle elongation also varied in rate but showed a different periodicity than translocation of the spindle, and therefore appeared independently regulated. The length of the spindle increased with the length of the cell, and the rate of spindle elongation and pole movement increased with distance moved, indicating that the forces mediated by astral microtubules increase with their length. Arp1/dynactin, not colocalising with tubulin, was more often continuous with microtubules in anaphase B than in metaphase, and was primarily located at the bottom of the cell. Further, shifts in the geometric gravity centre of the cell occurred in the same direction as migration of the spindle. To explain these results, we suggest that astral microtubles transiently anchored at the bottom of the cell are of particular importance for spindle translocation in fibroblasts.     

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.     

Latrunculin A delays anaphase onset in fission yeast by disrupting an Ase1-independent pathway controlling mitotic spindle stability

It has been proposed previously that latrunculin A, an inhibitor of actin polymerization, delays the onset of anaphase by causing spindle misorientation in fission yeast. However, we show that Deltamto1 cells, which are defective in nucleation of cytoplasmic microtubules, have profoundly misoriented spindles but are not delayed in the timing of sister chromatid separation, providing compelling evidence that fission yeast does not possess a spindle orientation checkpoint. Instead, we show that latrunculin A delays anaphase onset by disrupting interpolar microtubule stability. This effect is abolished in a latrunculin A-insensitive actin mutant and exacerbated in cells lacking Ase1, which cross-links antiparallel interpolar microtubules at the spindle midzone both before and after anaphase. These data indicate that both Ase1 and an intact actin cytoskeleton are required for preanaphase spindle stability. Finally, we show that loss of Ase1 activates a checkpoint that requires only the Mad3, Bub1, and Mph1, but not Mad1, Mad2, or Bub3 checkpoint proteins.     

A model for cleavage plane determination in early amphibian and fish embryos

Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.     

Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.     

Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance

Spindle orientation defines the plane of cell division and, thereby, the spatial position of all daughter cells. Here, we develop a live cell microscopy-based methodology to extract spindle movements in human epithelial cell lines and study how spindles are brought to a pre-defined orientation. We show that spindles undergo two distinct regimes of movements. Spindles are first actively rotated toward the cells’ long-axis and then maintained along this pre-defined axis. By quantifying spindle movements in cells depleted of LGN, we show that the first regime of rotational movements requires LGN that recruits cortical dynein. In contrast, the second regime of movements that maintains spindle orientation does not require LGN, but is sensitive to 2ME2 that suppresses microtubule dynamics. Our study sheds first insight into spatially defined spindle movement regimes in human cells, and supports the presence of LGN and dynein independent cortical anchors for astral microtubules.     

EB 1 immunofluorescence reveals an increase in growing astral microtubule length and number during anaphase in NRK-52E cells

Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.     

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.     

Mammalian spindle orientation and position respond to changes in cell shape in a dynein-dependent fashion

In animal cells, positioning of the mitotic spindle is crucial for defining the plane of cytokinesis and the size ratio of daughter cells. We have characterized this phenomenon in a rat epithelial cell line using microscopy, micromanipulation, and microinjection. Unmanipulated cells position the mitotic spindle near their geometric center, with the spindle axis lying roughly parallel to the long axis of the cell. Spindles that were initially misoriented underwent directed rotation and caused a delay in anaphase onset. To gain further insight into this process, we gently deformed cells with a blunted glass needle to change the spatial relationship between the cortex and spindle. This manipulation induced spindle movement or rotation in metaphase and/or anaphase, until the spindle reached a proper position relative to the deformed shape. Spindle positioning was inhibited by either treatment with low doses of nocodazole or microinjection of antibodies against dynein, apparently due to the disruption of the organization of dynein and/or astral microtubules. Our results suggest that mitotic cells continuously monitor and maintain the position of the spindle relative to the cortex. This process is likely driven by interactions among astral microtubules, the motor protein dynein, and the cell cortex and may constitute part of a mitotic checkpoint mechanism.     

Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner

The orientation of mitotic spindles is tightly regulated in polarized cells, but it has been unclear whether there is a mechanism regulating spindle orientation in nonpolarized cells. Here we show that integrin-dependent cell adhesion to the substrate orients the mitotic spindle of nonpolarized cultured cells parallel to the substrate plane. The spindle is properly oriented in cells plated on fibronectin or collagen, but misoriented in cells on poly-L-lysine or treated with the RGD peptide or anti-beta1-integrin antibody, indicating requirement of integrin-mediated cell adhesion for this mechanism. Remarkably, this mechanism is independent of gravitation or cell-cell adhesion, but requires actin cytoskeleton and astral microtubules. Furthermore, myosin X and the microtubule plus-end-tracking protein EB1 are shown to play a role in this mechanism through remodeling of actin cytoskeleton and stabilization of astral microtubules, respectively. Our results thus uncover the existence of a mechanism that orients the spindle parallel to the cell-substrate adhesion plane, and identify crucial factors involved in this novel mechanism.