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 self-association in Fin1 function on the mitotic spindle

Stabilization of spindle microtubules during anaphase is essential for proper chromosome segregation. Fin1 is a budding yeast protein that localizes to the poles and microtubules of the spindle during anaphase and contributes to spindle stability. The N-terminal half of Fin1 is phosphorylated at multiple sites by the cyclin-dependent kinase Clb5-Cdk1, and dephosphorylation in anaphase triggers its localization to the spindle. The C-terminal half of Fin1 contains coiled-coil motifs that are required for its self-association. Here we investigated the functional importance of the two regions of Fin1. Fin1 mutants lacking the C-terminal coiled-coil domains localized to spindle pole bodies but not along spindle microtubules. These mutants failed to self-associate and displayed reduced binding to microtubules in vitro but were functional in vivo and stabilized anaphase spindles when dephosphorylated. Deletion of the Fin1 C terminus suppressed the lethal phenotypes of the phospho-mutant Fin15A. Our findings suggest that the N-terminal region of Fin1 is sufficient for its regulated function as a spindle-stabilizing factor and that this function involves association with the spindle pole body. The ability of the C-terminal region to promote Fin1 self-association and microtubule binding may underlie the lethal effects of the deregulated Fin15A mutant.     

Spatial regulation improves antiparallel microtubule overlap during mitotic spindle assembly

The mitotic spindle plays an essential role in chromosome segregation during cell division. Spindle formation and proper function require that microtubules with opposite polarity overlap and interact. Previous computational simulations have demonstrated that these antiparallel interactions could be created by complexes combining plus- and minus-end-directed motors. The resulting spindles, however, exhibit sparse antiparallel microtubule overlap with motor complexes linking only a nominal number of antiparallel microtubules. Here we investigate the role that spatial differences in the regulation of microtubule interactions can have on spindle morphology. We show that the spatial regulation of microtubule catastrophe parameters can lead to significantly better spindle morphology and spindles with greater antiparallel MT overlap. We also demonstrate that antiparallel microtubule overlap can be increased by having new microtubules nucleated along the length of existing astral microtubules, but this increase negatively affects spindle morphology. Finally, we show that limiting the diffusion of motor complexes within the spindle region increases antiparallel microtubule interaction.     

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.     

Myosin-10 and actin filaments are essential for mitotic spindle function

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.     

Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization.     

The ran GTPase regulates mitotic spindle assembly.

Ran is an abundant nuclear GTPase with a clear role in nuclear transport during interphase but with roles in mitotic regulation that are less well understood. The nucleotide-binding state of Ran is regulated by a GTPase activating protein, RanGAP1, and by a guanine nucleotide exchange factor, RCC1. Ran also interacts with a guanine nucleotide dissociation inhibitor, RanBP1. RanBP1 has a high affinity for GTP-bound Ran, and it acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP hydrolysis on Ran approximately tenfold. RanBP1 levels oscillate during the cell cycle [4], and increased concentrations of RanBP1 prolong mitosis in mammalian cells and in Xenopus egg extracts (our unpublished observations). We investigated how increased concentrations of RanBP1 disturb mitosis. We found that spindle assembly is dramatically disrupted when exogenous RanBP1 is added to M phase Xenopus egg extracts. We present evidence that the role of Ran in spindle assembly is independent of nuclear transport and is probably mediated through changes in microtubule dynamics.     

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.     

The spindle protein CHICA mediates localization of the chromokinesin Kid to the mitotic spindle

Microtubule-based motor proteins provide essential forces for bipolar organization of spindle microtubules and chromosome movement, prerequisites of chromosome segregation during the cell cycle. Here, we describe the functional characterization of a novel spindle protein, termed "CHICA," that was originally identified in a proteomic survey of the human spindle apparatus [1]. We show that CHICA localizes to the mitotic spindle and is both upregulated and phosphorylated during mitosis. CHICA-depleted cells form shorter spindles and fail to organize a proper metaphase plate, highly reminiscent of the phenotype observed upon depletion of the chromokinesin Kid, a key mediator of polar ejection forces [2-6]. We further show that CHICA coimmunoprecipitates with Kid and is required for the spindle localization of Kid without affecting its chromosome association. Moreover, upon depletion of either CHICA or Kid (or both proteins simultaneously), chromosomes collapse onto the poles of monastrol-induced monopolar spindles. We conclude that CHICA represents a novel interaction partner of the chromokinesin Kid that is required for the generation of polar ejection forces and chromosome congression.     

The role of Cdc55 in the spindle checkpoint is through regulation of mitotic exit in Saccharomyces cerevisiae

Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete.     

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.     

External forces control mitotic spindle positioning

The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.     

A Lim protein involved in the progression of cytokinesis and regulation of the mitotic spindle

DdLimE regulates cell motility and cytokinesis in Dictyostelium. To specify its function, we generated knock-out mutants and analyzed mitosis by marking the mitotic apparatus with GFP-alpha-tubulin. Characteristic of DdLimE-null cells is a late reversal of cytokinesis caused by backward movement of the incipient daughter cells. This process of "retro-cytokinesis" is accompanied by a delay in disassembly of the mitotic spindle. The length of interphase microtubules is increased and their depolymerization at prophase is impaired. These data indicate that DdLimE links the cortical actin network, where it is located, to the microtubule system, whose dynamics it regulates.     

Centrosome-independent mitotic spindle formation in vertebrates

BACKGROUND: In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS: These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.     

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.