Cep192 and the generation of the mitotic spindle

The cellular mechanisms used to generate sufficient microtubule polymer mass to drive the assembly and function of the mitotic spindle remain a matter of great interest. As the primary microtubule nucleating structures in somatic animal cells, centrosomes have been assumed to figure prominently in spindle assembly. At the onset of mitosis, centrosomes undergo a dramatic increase in size and microtubule nucleating capacity, termed maturation, which is likely a key event in mitotic spindle formation. Interestingly, however, spindles can still form in the absence of centrosomes calling into question the specific mitotic role of these organelles. Recent work has shown that the human centrosomal protein, Cep192, is required for both centrosome maturation and spindle assembly thus providing a molecular link between these two processes. In this article, we propose that Cep192 does so by forming a scaffolding on which proteins involved in microtubule nucleation are sequestered and become active in mitotic cells. Normally, this activity is largely confined to centrosomes but in their absence continues to function but is dispersed to other sites within the cell.     

Spindle assembly requires complete disassembly of spindle remnants from the previous cell cycle

Incomplete mitotic spindle disassembly causes lethality in budding yeast. To determine why spindle disassembly is required for cell viability, we used live-cell microscopy to analyze a double mutant strain containing a conditional mutant and a deletion mutant compromised for the kinesin-8 and anaphase-promoting complex-driven spindle-disassembly pathways (td-kip3 and doc1Δ, respectively). Under nonpermissive conditions, spindles in td-kip3 doc1Δ cells could break apart but could not disassemble completely. These cells could exit mitosis and undergo cell division. However, the daughter cells could not assemble functional, bipolar spindles in the ensuing mitosis. During the formation of these dysfunctional spindles, centrosome duplication and separation, as well as recruitment of key midzone-stabilizing proteins all appeared normal, but microtubule polymerization was nevertheless impaired and these spindles often collapsed. Introduction of free tubulin through episomal expression of α- and β-tubulin or introduction of a brief pulse of the microtubule-depolymerizing drug nocodazole allowed spindle assembly in these td-kip3 doc1Δ mutants. Therefore we propose that spindle disassembly is essential for regeneration of the intracellular pool of assembly-competent tubulin required for efficient spindle assembly during subsequent mitoses of daughter cells.     

Multiple Cancer Testis Antigens Function To Support Tumor Cell Mitotic Fidelity

While the expression of genes that are normally involved in spermatogenesis is frequently detected in tumors, the extent to which these gene products are required for neoplastic behaviors is unclear. To begin to address their functional relevance to tumorigenesis, we identified a cohort of proteins which display synthetic lethality with paclitaxel in non-small-cell lung cancer and whose expression is biased toward testes and tumors. Remarkably, these testis proteins, FMR1NB, NXF2, MAGEA5, FSIP1, and STARD6, are required for accurate chromosome segregation in tumor cells. Their individual depletion enhances the generation of multipolar spindles, increases mitotic transit time, and induces micronucleation in response to an otherwise innocuous dose of paclitaxel. The underlying basis for abnormal mitosis is an alteration in microtubule function, as their depletion increases microtubule cytaster formation and disrupts microtubule stability. Given these observations, we hypothesize that reactivated testis proteins may represent unique tumor cell vulnerabilities which, if targeted, could enhance responsiveness to antimitotic therapy. Indeed, we demonstrate that combining paclitaxel with a small-molecule inhibitor of the gametogenic and tumor cell mitotic protein TACC3 leads to enhanced centrosomal abnormalities, activation of death programs, and loss of anchorage-independent growth.     

A unique insertion in STARD9's motor domain regulates its stability

STARD9 is a largely uncharacterized mitotic kinesin and putative cancer target that is critical for regulating pericentriolar material cohesion during bipolar spindle assembly. To begin to understand the mechanisms regulating STARD9 function and their importance to cell division, we took a multidisciplinary approach to define the cis and trans factors that regulate the stability of the STARD9 motor domain. We show that, unlike the other ∼50 mammalian kinesins, STARD9 contains an insertion in loop 12 of its motor domain (MD). Working with the STARD9-MD, we show that it is phosphorylated in mitosis by mitotic kinases that include Plk1. These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation. Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects. Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.     

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.     

New frontiers: discovering cilia‐independent functions of cilia proteins

In most vertebrates, mitotic spindles and primary cilia arise from a common origin, the centrosome. In non‐cycling cells, the centrosome is the template for primary cilia assembly and, thus, is crucial for their associated sensory and signaling functions. During mitosis, the duplicated centrosomes mature into spindle poles, which orchestrate mitotic spindle assembly, chromosome segregation, and orientation of the cell division axis. Intriguingly, both cilia and spindle poles are centrosome‐based, functionally distinct structures that require the action of microtubule‐mediated, motor‐driven transport for their assembly. Cilia proteins have been found at non‐cilia sites, where they have distinct functions, illustrating a diverse and growing list of cellular processes and structures that utilize cilia proteins for crucial functions. In this review, we discuss cilia‐independent functions of cilia proteins and re‐evaluate their potential contributions to “cilia” disorders.     

Live-cell analysis of mitotic spindle formation in taxol-treated cells

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.     

Heat shock alters centrosome organization leading to mitotic dysfunction and cell death

To identify the cellular target(s) responsible for thermal killing in the G₁ phase of the cell cycle, synchronous cultures of Chinese hamster ovary cells (CHO) were heat shocked and studied for one cell cycle by time-lapse videomicroscopy and immunocytochemistry. At the first mitosis post-heating, the fraction of cells giving rise to multinucleated progeny approximately equaled the nonclonogenic fraction. In addition, the cells yielding multincleated progeny were delayed in prophase-metaphase relative to the cells yielding two uninucleated progeny (clonogenic cells). To study the basis for the delay in prophase-metaphase and subsequent formation of multinucleated cells, cells in mitosis were examined by immunofluorescence for spindle abnormalities. Multipolar mitotic spindles and chromosome misalignment were induced by heat. All multiple spindle poles induced by heat stained for pericentriolar material (PCM), the microtubule nucleating material of centrosomes. Heated cells in mitosis also contained additional foci of PCM which were not associated with the spindle. Cells made thermotolerant by a nonlethal heat shock were resistant to both thermal killing and the induction of multiple foci of PCM. Quantitative analysis revealed a good correlation between the fraction of cells with multipolar spindles, the fraction with more than two foci of PCM, and the nonclonogenic fraction. These data indicate that heat-induced alterations to the PCM of centrosomes resulted in multipolar mitotic spindles, delay in prophase-metaphase, and formation of multinucleated cells which were nonclonogenic. These results identify the centrosome as a G₁ target for cell killing. © 1993 Wiley-Liss, Inc.     

The centrosome and bipolar spindle assembly: Does one have anything to do with the other?

In vertebrate somatic cells, the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated. In naturally occurring cellular systems that lack centrioles, such as plant cells and many oocytes, bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization, both before and after nuclear envelope breakdown (NEB), is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.Key words: centrosome, centriole, mitosis, spindle, cell cycle, meiosis, plant cell, microsurgery     

Drosophila EB1 is important for proper assembly,dynamics, and positioning of the mitotic spindle

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.     

Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.     

The role of stathmin in the regulation of the cell cycle

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.     

Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.     

Structural and regulatory roles of nonmotor spindle proteins

Chromosome alignment and segregation during cell division rely on a highly ordered bipolar microtubule array called the mitotic spindle. The organization of microtubules into bipolar spindles with focused poles during mitosis requires numerous microtubule-associated proteins including both motor and nonmotor proteins. Nonmotor microtubule-associated proteins display extraordinary diversity in how they contribute to mitotic spindle organization. These mechanisms include regulation of microtubule nucleation and organization, direct and indirect influences on motor function, and control of cell cycle progression. Furthermore, many nonmotor spindle proteins display altered expression in cancer cells emphasizing their important roles in cell proliferation.     

Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.     

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.     

Kinesin-5 inhibitor resistance is driven by kinesin-12

The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼10⁷ cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process.     

A kinase-independent role for Aurora A in the assembly of mitotic spindle microtubules in Caenorhabditis elegans embryos

The assembly of a functional mitotic spindle is crucial for achieving successful mitosis. Aurora A kinase is one of the key regulators of mitotic events, including mitotic entry, centrosome maturation and spindle bipolarity. Caenorhabditis elegans Aurora A (AIR-1) is responsible for the assembly of γ-tubulin-independent microtubules in early embryos; however, the mechanism by which AIR-1 contributes to microtubule assembly during mitosis has been unclear. Here we show by live-cell imaging and RNA-mediated interference (RNAi)-based modulation of gene activity that AIR-1 has a crucial role in the assembly of chromatin-stimulated microtubules that is independent of the γ-tubulin complex. Surprisingly, the kinase activity of AIR-1 is dispensable for this process. Although the kinase-inactive form of AIR-1 was detected along the microtubules as well as on centrosomes, the kinase-active form of AIR-1 was restricted to centrosomes. Thus, we propose that AIR-1 has a kinase-dependent role at centrosomes and a kinase-independent role for stabilizing spindle microtubules and that coordination of these two roles is crucial for the assembly of mitotic spindles.     

The centrosome and bipolar spindle assembly

In vertebrate somatic cells the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated. In naturally occurring cellular systems that lack centrioles - such as plant cells and many oocytes - bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them, and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization- both before and after nuclear envelope breakdown (NEB) - is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.     

Survivin modulates microtubule dynamics and nucleation throughout the cell cycle

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.