Length control of the metaphase spindle

BACKGROUND: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. RESULTS: Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. CONCLUSIONS: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.     

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.     

TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux.     

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 chromokinesin Kid is required for maintenance of proper metaphase spindle size

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.     

Mechanisms of spindle assembly and size control

The spindle is crucial for cell division by allowing the faithful segregation of replicated chromosomes to daughter cells. Proper segregation is ensured only if microtubules (MTs) and hundreds of other associated factors interact to assemble this complex structure with the appropriate architecture and size. In this review, we describe the latest view of spindle organisation as well as the molecular gradients and mechanisms underlying MT nucleation and spindle assembly. We then discuss the overlapping physical and molecular constraints that dictate spindle morphology, concluding with a focus on spindle size regulation.     

RAB-11 permissively regulates spindle alignment by modulating metaphase microtubule dynamics in Caenorhabditis elegans early embryos

Alignment of the mitotic spindle along a preformed axis of polarity is crucial for generating cell diversity in many organisms, yet little is known about the role of the endomembrane system in this process. RAB-11 is a small GTPase enriched in recycling endosomes. When we depleted RAB-11 by RNAi in Caenorhabditis elegans, the spindle of the one-cell embryo failed to align along the axis of polarity in metaphase and underwent violent movements in anaphase. The distance between astral microtubules ends and the anterior cortex was significantly increased in rab-11(RNAi) embryos specifically during metaphase, possibly accounting for the observed spindle alignment defects. Additionally, we found that normal ER morphology requires functional RAB-11, particularly during metaphase. We hypothesize that RAB-11, in conjunction with the ER, acts to regulate cell cycle-specific changes in astral microtubule length to ensure proper spindle alignment in Caenorhabditis elegans early embryos.     

Effect of metabolic inhibitors on sucrose-induced metaphase spindle elongation and spindle recovery

Hyperosmotic sucrose treatment of metaphase PtK-1 cells has been shown to produce a reversible concentration-dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366-372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62-69, 1984 and 39:373-379, 1985). Addition of metabolic inhibitors to Ham's F-12 salts with deoxyglucose (D/F-12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose-induced spindle elongation; rates and extents are 60-75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F-12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose-treated cells released into D/F-12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP-containing D/F-12 medium and released into DNP-containing D/F-12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose-induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.     

Ran is required before metaphase for spindle assembly and chromosome alignment and after metaphase for chromosome segregation and spindle midbody organization

The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.     

The spindle checkpoint protein Mad2 regulates APC/C activity during prometaphase and metaphase of meiosis I in Saccharomyces cerevisiae

In many eukaryotes, disruption of the spindle checkpoint protein Mad2 results in an increase in meiosis I nondisjunction, suggesting that Mad2 has a conserved role in ensuring faithful chromosome segregation in meiosis. To characterize the meiotic function of Mad2, we analyzed individual budding yeast cells undergoing meiosis. We find that Mad2 sets the duration of meiosis I by regulating the activity of APC(Cdc20). In the absence of Mad2, most cells undergo both meiotic divisions, but securin, a substrate of the APC/C, is degraded prematurely, and prometaphase I/metaphase I is accelerated. Some mad2Δ cells have a misregulation of meiotic cell cycle events and undergo a single aberrant division in which sister chromatids separate. In these cells, both APC(Cdc20) and APC(Ama1) are prematurely active, and meiosis I and meiosis II events occur in a single meiotic division. We show that Mad2 indirectly regulates APC(Ama1) activity by decreasing APC(Cdc20) activity. We propose that Mad2 is an important meiotic cell cycle regulator that ensures the timely degradation of APC/C substrates and the proper orchestration of the meiotic divisions.     

MscS-like proteins control plastid size and shape in Arabidopsis thaliana

BACKGROUND: Mechanosensitive (MS) ion channels provide a mechanism for the perception of mechanical stimuli such as sound, touch, and osmotic pressure. The bacterial MS ion channel MscS opens in response to increased membrane tension and serves to protect against cellular lysis during osmotic downshock. MscS-like proteins are found widely in bacterial and archaeal species and have also been identified in fission yeast and plants. None of the eukaryotic members of the family have yet been characterized. RESULTS: Here, we characterize two MscS-like (MSL) proteins from Arabidopsis thaliana, MSL2 and MSL3. MSL3 can rescue the osmotic-shock sensitivity of a bacterial mutant lacking MS-ion-channel activity, suggesting that it functions as a mechanosensitive ion channel. Arabidopsis plants harboring insertional mutations in both MSL3 and MSL2 show abnormalities in the size and shape of plastids, which are plant-specific endosymbiotic organelles responsible for photosynthesis, gravity perception, and numerous metabolic reactions. MSL2-GFP and MSL3-GFP are localized to discrete foci on the plastid envelope and colocalize with the plastid division protein AtMinE. CONCLUSIONS: Our data support a model wherein MSL2 and MSL3 control plastid size, shape, and perhaps division during normal plant development by altering ion flux in response to changes in membrane tension. We propose that MscS family members have evolved new roles in plants since the endosymbiotic event that gave rise to plastids.     

Asymmetric division of spindle microtubules and microfilaments during bovine meiosis from metaphase I to metaphase III

The kinetics of spindle and chromosomes during bovine oocyte meiosis from meiosis I to meiosis III is described. The results of this study showed that (1) oocytes began to extrude the first polar body (Pb1) at the early anaphase I stage and the Pb1 totally separated from the mother cell only when oocytes reach the MII stage; (2) the morphology of the spindle changed from barrel-shaped at the metaphase stage to cylinder-shaped at early anaphase, and then to a thin, long triangle-shaped cone at late anaphase and telophase stages; (3) chromosome morphology went from an individual visible stage at metaphase to a less defined chromatin state during anaphase and telophase stages, and then back to visible individual chromosomes at the next metaphase; (4) chromatin that connected with the floor of the cone became the polar bodies and expelled, and almost all of the microtubules (MTs) and microfilaments (MFs) composing the spindles moved towards and contributed to the polar bodies; and (5) the size of the metaphase I (MI) spindle was larger than the metaphase II (MII) and metaphase III (MIII) spindles. The MII spindle, however, is more barrel-shaped than the MI spindle. This study suggests that spindle MTs and MFs during bovine oocyte meiosis are asymmetrically divided into the polar bodies.     

Mechanisms of microtubule-based kinetochore positioning in the yeast metaphase spindle

It has been hypothesized that spatial gradients in kMT dynamic instability facilitate mitotic spindle formation and chromosome movement. To test this hypothesis requires the analysis of kMT dynamics, which have not been resolved at the single kMT level in living cells. The budding yeast spindle offers an attractive system in which to study kMT dynamics because, in contrast to animal cells, there is only one kMT per kinetochore. To visualize metaphase kMT plus-end dynamics in yeast, a strain containing a green fluorescent protein fusion to the kinetochore protein, Cse4, was imaged by fluorescence microscopy. Although individual kinetochores were not resolvable, we found that models of kMT dynamics could be evaluated by simulating the stochastic kMT dynamics and then simulating the fluorescence imaging of kMT plus-end-associated kinetochores. Statistical comparison of model-predicted images to experimentally observed images demonstrated that a pure dynamic instability model for kMT dynamics in the yeast metaphase spindle was unacceptable. However, when a temporally stable spatial gradient in the catastrophe or rescue frequency was added to the model, there was reasonable agreement between the model and the experiment. These results provide the first evidence of temporally stable spatial gradients of kMT catastrophe and/or rescue frequency in living cells.