The regulation of mitotic spindle function

The process of mitosis includes a series of morphological changes in the cell in which the directional movements of chromosomes are the most prominent. The presence of a microtubular array, known as the spindle or mitotic apparatus, provides at least a scaffold upon which these movements take place. The precise mechanism for chromosome movement remains obscure, but new findings suggest that the kinetochore may play a key role in chromosome movement toward the spindle pole, and that sliding interactions between or among adjacent microtubules may provide the mechanochemical basis for spindle elongation. The physiological regulation of the anaphase motors and of spindle operation either before or after anaphase remains equally elusive. Elicitors that may serve as controlling elements in spindle function include shifts in cytosolic calcium activity and perhaps the activation or inactivation of protein kinases, which in turn produce changes in the state of phosphorylation of specific spindle components.     

Dynactin function in mitotic spindle positioning

Dynactin is a multisubunit protein complex necessary for dynein function. Here, we investigated the function of dynactin in budding yeast. Loss of dynactin impaired movement and positioning of the mitotic spindle, similar to loss of dynein. Dynactin subunits required for function included p150^Glued, dynamitin, actin-related protein (Arp) 1 and p24. Arp10 and capping protein were dispensable, even in combination. All dynactin subunits tested localized to dynamic plus ends of cytoplasmic microtubules, to stationary foci on the cell cortex and to spindle pole bodies. The number of molecules of dynactin in those locations was small, less than five. In the absence of dynactin, dynein accumulated at plus ends and did not appear at the cell cortex, consistent with a role for dynactin in offloading dynein from the plus end to the cortex. Dynein at the plus end was necessary for dynactin plus-end targeting. p150^Glued was the only dynactin subunit sufficient for plus-end targeting. Interactions among the subunits support a molecular model that resembles the current model for brain dynactin in many respects; however, three subunits at the pointed end of brain dynactin appear to be absent from yeast.     

The role of localization in the operation of the mitotic spindle assembly checkpoint

The mitotic spindle assembly checkpoint (^MSAC) is an important regulatory mechanism of the cell cycle, ensuring proper chromosome segregation in mitosis. It delays the transition to anaphase until all chromosomes are properly attached to the mitotic spindle by emitting a diffusible “wait anaphase”-signal from unattached kinetochores. Current models of the checkpoint disregard important spatial properties like localization, diffusion and realistic numbers of kinetochores. To allow for in silico studies of the dynamics of these models in a more realistic environment, we introduce a mathematical framework for quasi-spatial simulation of localized biochemical processes that are typically observed during checkpoint activation and maintenance. The “emitted inhibition” model of the ^MSAC by Doncic et al. (Proc Natl Acad Sci USA 2005; 102:6332–7) assumes instantaneous activation of the diffusible “wait anaphase”-signal upon kinetochore encounter. We modify this model to account for binding kinetics with finite rates and use the developed framework to determine the feasible range of the binding parameters. We find that for proper activation, the binding rate constant has to be fast and above a critical value. Furthermore, this critical value depends significantly on the amount of local binding sites at each kinetochore. The critical values lie in a physiological realistic regime (10⁴–10⁶ M^-1s^-1). We also determine the feasible parameter range for fast checkpoint activation of the “Mad2 template” model, for which the kinetic parameters have recently been studied in vitro by Simonetta et al. (PLoS Biology 2009; 7:1000010). We find critical values for binding and catalysis rate constants, both significantly higher than the measured values. Our results suggest that yet unknown mechanisms at the kinetochores facilitate binding and catalysis in vivo. We conclude that quantitative models of the ^MSAC have to account for the limited availability of binding sites at kinetochores.     

Concentrating on the mitotic spindle

In eukaryotes, the microtubule-based spindle drives chromosome segregation. In this issue, Schweizer et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506107) find that the spindle area is demarcated by a semipermeable organelle barrier. Molecular crowding, which is microtubule independent, causes the enrichment and/or retention of crucial factors in the spindle region. Their results add an important new feature to the models of how this structure assembles and is regulated.Mitosis is marked by the assembly of the mitotic spindle, a microtubule-based structure that facilitates accurate chromosome segregation. Many biochemical reactions are coupled to spindle assembly, from tubulin polymerization itself to the mitotic checkpoint, which inhibits chromosome disjunction until all the chromosomes are properly attached and aligned (Cleveland et al., 2003). Interestingly, these reactions are virtually unaltered over a broad morphological range; large spindles and small spindles follow roughly the same biochemical rules despite quite distinct geometries (Brown et al., 2007; Wühr et al., 2008). In this issue, Schweizer et al. report that the mitotic spindle area is delineated by membrane-bound organelles, generating a “spindle envelope” with unique molecular constituents compared with the surrounding cytoplasm. Spindle envelope–based molecular crowding provides an enticing hypothetical solution to the broad problem of confining mitotic biochemistry to a specific cellular space irrespective of cell size.Schweizer et al. (2015) used FRAP and fluorescence correlation spectroscopy (FCS) to measure the mobility of specific proteins. The authors found that tubulin and the Mad2 spindle assembly checkpoint protein were enriched in the spindle area in a microtubule polymer-independent manner. Given that the mobility of these proteins outside and within the spindle area was the same, changes in local concentration were likely a result of a barrier effect. In support of this hypothesis, modeling their FCS data with a fenestrated barrier separating the spindle area from the cytoplasm reproduced the measured FCS results. To test if a barrier surrounding the spindle area was important for spindle function, the authors disrupted the envelope area by laser microsurgery and found chromosome segregation errors consistent with defects in spindle assembly and kinetochore attachment monitoring. Thus, spindle envelope–based concentration of basal components in two critical spindle reactions, spindle assembly and mitotic checkpoint signaling, could mechanistically catalyze cell division.Molecular crowding can catalyze reactions and stabilize proteins by altering the local concentration of one or more rate-limiting components and is best characterized by membrane-bound organelles. Crowding by aggregation can greatly increase biochemical reactions without the need for a contiguous membrane (Weber and Brangwynne, 2012; Brangwynne, 2013) and this phenomenon can regulate cell cycle states (Lee et al., 2013). Here, Schweizer et al. (2015) propose that by creating a membrane-bound organelle exclusion zone, a spindle envelope could cause the molecular crowding of important spindle proteins and thereby their enrichment in the spindle area.The mitotic spindle is known to scale with cell size: smaller cells have smaller spindles (Levy and Heald, 2012). Spindle size scaling is prominent during development when repeated cell division without embryonic growth results in cells that can be several orders of magnitude smaller than that of the zygote. Recently, cytoplasm volume and tubulin concentration was shown to be an important factor in spindle size scaling; however, a curious exception to the size scaling rule is that there seems to be an upper limit to spindle size, resulting in stable spindle size when a threshold cell size is reached (Wühr et al., 2008; Good et al., 2013; Hazel et al., 2013). A spindle envelope would provide mechanisms to maintain increased local tubulin concentration independent of the absolute amount available in the cell. The net effect would be that spindle size scales in very large cells to the spindle envelope size rather than cell size in a manner analogous to chromosome size scaling to nuclear size independently of cell size (Fig. 1 A). Clearly this is a more complex problem and factors such as tubulin protein production and polymerization cofactors (such as the Tog family of proteins) clearly play an important role (Slep, 2009). However, spindle envelope–based molecular crowding could provide an elegant solution to a biochemical problem.Open in a separate windowFigure 1.Molecular crowding in the mitotic spindle. Schematic view of how a spindle envelope could mitigate spindle size scaling during development (A) or cell cycle control (B) in a common cytoplasm. (A) A spindle envelope (black dotted line) that excludes large membrane-bound organelles (yellow) could locally increase the concentration of spindle proteins (depicted as a red background) such as tubulin to control spindle size independent of cell size during early development. (B) Neighboring nuclei in a common cytoplasm (e.g., the syncytial mitotic gonad in C. elegans) could have differing mitotic states by restricting the diffusion of important regulatory proteins such as Mad2 (similar coloring as in A).A spindle envelope could also provide a cell biological solution to another developmental problem—independent cell cycle control of separate nuclei within a single cytoplasm (syncytia). For example, the mitotic region of the Caenorhabditis elegans germline contains germ cell precursors that divide independently of one another in a common cytoplasm. In some cases, two neighboring dividing nuclei can have different biochemistry, one arrested in metaphase because of a kinetochore microtubule attachment defect while the other is progressing into anaphase (Gerhold et al., 2015). By restricting the diffusive radius of signaling molecules like Mad2, a steep threshold of checkpoint activity can be maintained, allowing independent cell cycle control even in a common cytoplasm (Fig. 1 B).The mitotic spindle has long been known to exclude large membrane-bound organelles, even in the absence of microtubule polymer, leading to a hypothesized nontubulin-based “spindle matrix.” A spindle matrix would be an excellent candidate to underlie the spindle envelope. The molecular nature of a spindle matrix, however, has never been agreed upon with candidate mechanisms ranging from nonprotein macromolecules to actin (Pickett-Heaps et al., 1984; Chang et al., 2004). A convincing argument can be made that the Skeletor/Megator/Chromator proteins first identified in Drosophila melanogaster constitute a spindle matrix (Walker et al., 2000; Qi et al., 2004; Rath et al., 2004; Schweizer et al., 2014). These proteins are large and are found in the nucleus in interphase and as microtubule-independent fibrous structures in and around the spindle in mitosis. Depletion of these proteins results in mitotic errors; however, these may or may not be caused by a role as the spindle matrix.Schweizer et al. (2015) evaluated Megator (as a representative member of the complex) as a possible basis for generating the spindle envelope. FRAP and FCS showed that, like tubulin and Mad2, Megator is concentrated in the spindle envelope region independent of microtubules. However, Megator in the spindle region had slower diffusive properties compared with that around the cell periphery. Thus, unlike tubulin and Mad2, the mobility of Megator within the spindle was altered, indicating that Megator likely forms a high molecular weight complex with its binding partners Skeletor and Chromator in the spindle area, which may help form the spindle envelope.The sum of these results lead to a possible model whereby the Skeletor/Megator/Chromator proteins complex together and subsequently support a spindle envelope independent of microtubules. The spindle envelope excludes large membrane-bound organelles, leading to increased concentration of mitotic reaction constituents and thus ultimately catalyzing cell division. It will be exciting in the future to determine if the spindle area is indeed subject to molecular crowding in the purest of forms (solvent exclusion) and how this effect drives cell division.     

Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function

The mitotic exit network (MEN) is a signaling cascade that triggers inactivation of the mitotic cyclin-dependent kinases and exit from mitosis. The GTPase Tem1 localizes on the spindle pole bodies (SPBs) and initiates MEN signaling. Tem1 activity is inhibited until anaphase by Bfa1-Bub2. These proteins are also part of the spindle position checkpoint (SPOC), a surveillance mechanism that restrains mitotic exit until the spindle is correctly positioned. Here, we show that regulation of Tem1 localization is essential for the proper function of the MEN and the SPOC. We demonstrate that the dynamics of Tem1 loading onto SPBs determine the recruitment of other MEN components to this structure, and reevaluate the interdependence in the localization of Tem1, Bfa1, and Bub2. We also find that removal of Tem1 from the SPBs is critical for the SPOC to impede cell cycle progression. Finally, we demonstrate for the first time that localization of Tem1 to the SPBs is a requirement for mitotic exit.     

Sucrose-induced spindle elongation in mitotic PtK-1 cells

Brief treatment of mitotic metaphase and anaphase PtK-1 cells with tissue culture medium containing 0.5 M sucrose resulted in spindle elongation without chromosome motion. Spindle birefringence also changed from a uniform appearance to one of highly birefringent bundles. Electron microscopic analysis indicated these birefringent bundles were composed of tightly packed arrays of spindle microtubules. No kinetochores could be seen following a 10 min sucrose treatment. Upon removal of sucrose, metaphase spindles returned to pretreatment lengths and the normal birefringence pattern returned. Reduction in spindle length could be temporally coupled with the reappearance of kinetochores and the reassociation of microtubules with these structures. In contrast to treated and released metaphase cells, anaphase spindles did not return to pretreatment lengths. Replacement of sucrose with medium showed the resumption of chromosome-to-pole motion within 2 min of sucrose removal. Chromosome motion could be correlated with the reappearance of kinetochores and kinetochore microtubules. These data have led us to postulate the existence of two microtubule continuums in the spindle and to discuss their roles in spindle organization and chromosome motion.     

Bi-orienting chromosomes on the mitotic spindle

For the proper segregation of sister chromatids before cell division, each sister kinetochore must attach to microtubules that extend to opposite spindle poles. This process is called bipolar microtubule attachment or chromosome bi-orientation. The mechanism for chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent studies suggest that cells can promote bi-orientation by re-orienting kinetochore-spindle pole connections.     

Chromosome bi-orientation on the mitotic spindle

For proper chromosome segregation, sister kinetochores must attach to microtubules extending from opposite spindle poles prior to anaphase onset. This state is called sister kinetochore bi-orientation or chromosome bi-orientation. The mechanism ensuring chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent evidence suggests that mal-oriented kinetochore-to-pole connections are corrected in a tension-dependent mechanism. The cohesin complex and the Ipl1/Aurora B protein kinase seem to be key regulators for this correction. In this article, I discuss how cells ensure sister kinetochore bi-orientation for all chromosomes, mainly focusing on our recent findings in budding yeast.     

Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.

We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.     

Cytoplasmic dynein plays a role in mammalian mitotic spindle formation

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP- hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.     

The mitotic spindle and actin tails

To segregate their chromosomes, eukaryotic cells rely on a dynamic structure made of microtubules: the mitotic spindle. This structure can form in cells lacking centrosomes, because their chromosomes also nucleate microtubules. This second assembly pathway is observed even in some cells that naturally have centrosomes, for example when the centrosomes are ablated by laser surgery. Recent results have started to address the complementary question of whether centrosome-nucleated microtubules alone could sustain the formation of a functional mitotic spindle. We wonder in this respect whether lower eukaryotes such as yeasts are different from higher eukaryotes such as vertebrates.     

The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly

The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.     

Quinacrine-induced changes in mitotic PtK1 spindle microtubule organization

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has been used to study the role of ATP requiring molecules in microtubule organization in mitotic PtK1 cells. Brief treatments of metaphase cells with concentrations of quinacrine ranging from 2 to 10 microM decreased spindle length and birefringence in a concentration-dependent manner. With either increasing quinacrine concentrations or duration of treatment, metaphase cells demonstrated a specific reorganization of spindle microtubules. Both polarization and electron microscopy showed a substantial loss of non-kinetochore spindle microtubules with an increase in astral microtubules: this was particularly evident in the region adjacent to the spindle domain. Addition of millimolar concentrations of dinitrophenol to quinacrine-containing medium did not potentiate the response of metaphase cells to quinacrine treatment. Time-lapse video analysis demonstrated that the astral microtubules are the result of reorganization of spindle microtubules. These data suggest that functional ATP binding sites are required to maintain stable interactions between microtubules and that these interactions are responsible for maintaining the bowed configuration of non-kinetochore spindle microtubules which are under compression at metaphase.     

A potential role for human cohesin in mitotic spindle aster assembly

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to form in vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.     

The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events

Centrosomes of vertebrate cells and spindle pole bodies (SPBs) of fungi were first recognized through their ability to organize microtubules. Recent studies suggest that centrosomes and SPBs also have a function in the regulation of cell cycle progression, in particular in controlling late mitotic events. Regulators of mitotic exit and cytokinesis are associated with the SPB of budding and fission yeast. Elucidation of the molecular roles played by these regulators is helping to clarify the function of the SPB in controlling progression though mitosis.