The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells

Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase-anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 microM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.     

CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.     

Still entangled: Assembly of the central spindle by multiple microtubule modulators

The central spindle is a microtubule-based structure that assembles during anaphase of mitosis in animal cells and is essential for multiple steps of cytokinesis. Central spindle assembly occurs by the cooperative action of multiple microtubule motors and modulators. Here, we review the mechanism by which the central spindle is formed, the role of several key proteins in this process and how central spindle assembly is temporally and spatially coordinated with mitosis.     

Centrosome/Spindle Pole–associated Protein Regulates Cytokinesis via Promoting the Recruitment of MyoGEF to the Central Spindle

Cooperative communications between the central spindle and the contractile ring are critical for the spatial and temporal regulation of cytokinesis. Here we report that MyoGEF, a guanine nucleotide exchange factor that localizes to the central spindle and cleavage furrow, interacts with centrosome/spindle pole-associated protein (CSPP), which is concentrated at the spindle pole and central spindle during mitosis and cytokinesis. Both in vitro and in vivo pulldown assays show that MyoGEF interacts with CSPP. The C-terminus of MyoGEF and N-terminus of CSPP are required for their interaction. Immunofluorescence analysis indicates that MyoGEF and CSPP colocalize at the central spindle. Depletion of CSPP or MyoGEF by RNA-interference (RNAi) not only causes defects in mitosis and cytokinesis, such as metaphase arrest and furrow regression, but also mislocalization of nonmuscle myosin II with a phosphorylated myosin regulatory light chain (p-MRLC). Importantly, CSPP depletion by RNAi interferes with MyoGEF localization at the central spindle. Finally, MyoGEF interacts with ECT2, and RNAi-mediated depletion of MyoGEF leads to mislocalization of ECT2 and RhoA during cytokinesis. Therefore, we propose that CSPP interacts with and recruits MyoGEF to the central spindle, where MyoGEF contributes to the spatiotemporal regulation of cytokinesis.     

Phosphorylation of ZEN-4/MKLP1 by aurora B regulates completion of cytokinesis

The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis [1]. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules [2, 3], and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly [4]. Centralspindlin localization depends on aurora B kinase [5]. Aurora B concentrates in the midbody and persists between daughter cells. Here, we show that in C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis.     

Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2

Mitotic kinases of the Polo and Aurora families are key regulators of chromosome segregation and cytokinesis. Here, we have investigated the role of MKlp1 and MKlp2, two vertebrate mitotic kinesins essential for cytokinesis, in the spatial regulation of the Aurora B kinase. Previously, we have demonstrated that MKlp2 recruits Polo-like kinase 1 (Plk1) to the central spindle in anaphase. We now find that in MKlp2 but not MKlp1-depleted cells the Aurora B-INCENP complex remains at the centromeres and fails to relocate to the central spindle. MKlp2 exerts dual control over Aurora B localization, because it is a binding partner for Aurora B, and furthermore for the phosphatase Cdc14A. Cdc14A can dephosphorylate INCENP and may contribute to its relocation to the central spindle in anaphase. We propose that MKlp2 is involved in the localization of Plk1, Aurora B, and Cdc14A to the central spindle during anaphase, and that the integration of signaling by these proteins is necessary for proper cytokinesis.     

Uncoupling the central spindle-associated function of the chromosomal passenger complex from its role at centromeres

Survivin is a component of the chromosomal passenger complex (CPC) that plays a role in maintenance of an active spindle checkpoint and in cytokinesis. To study whether these different functions can be attributed to distinct domains within the Survivin protein, we complemented Survivin-depleted cells with a variety of point- and deletion-mutants of Survivin. We show that an intact baculovirus IAP repeat (BIR) domain is required for proper spindle checkpoint functioning, but dispensable for cytokinesis. In line with this, mutants lacking an intact BIR domain localized normally to the central spindle, but their localization to inner centromeres was severely perturbed. Consequently, these mutants failed to recruit Aurora B, Borealin/Dasra B, and BubR1 to centromeres and kinetochores, but they had retained the ability to recruit Aurora B and Borealin/Dasra B to the midzone and midbody. Thus, the C terminus of Survivin is sufficient for central spindle localization and execution of cytokinesis, but the additional presence of a functional BIR domain is essential for centromere targeting and spindle checkpoint function. Importantly, our data show that the function of the CPC at the centromere can be separated from its function at the central spindle and that execution of cytokinesis does not require prior concentration of the CPC at centromeres.     

Drosophila male meiosis as a model system for the study of cytokinesis in animal cells

Drosophila male meiosis offers unique opportunities for mutational dissection of cytokinesis. This system allows easy and unambiguos identification of mutants defective in cytokinesis through the examination of spermatid morphology. Moreover, cytokinesis defects and protein immunostaining can be analyzed with exquisite cytological resolution because of the large size of meiotic spindles. In the past few years several mutations have been isolated that disrupt meiotic cytokinesis in Drosophila males. These mutations specify genes required for the assembly, proper constriction or disassembly of the contractile ring. Molecular characterization of these genes has identified essential components of the cytokinetic machinery, highlighting the role of the central spindle during cytokinesis. This structure appears to be both necessary and sufficient for signaling cytokinesis. In addition, many data indicate that the central spindle microtubules cooperatively interact with elements of the actomyosin contractile ring, so that impairment of either of these structures prevents the formation of the other.     

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.     

The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.     

PRC1 controls spindle polarization and recruitment of cytokinetic factors during monopolar cytokinesis

The central spindle is a postanaphase array of microtubules that plays an essential role in organizing the signaling machinery for cytokinesis. The model by which the central spindle organizes the cytokinetic apparatus is premised on an antiparallel arrangement of microtubules, yet cells lacking spindle bipolarity are capable of generating a distal domain of ectopic furrowing when forced into mitotic exit. Because protein regulator of cytokinesis (PRC1) and kinesin family member 4A (KIF4A) are believed to play a principal role in organizing the antiparallel midzone array, we sought to clarify their roles in monopolar cytokinesis. Although both factors localized to the distal ends of microtubules during monopolar cytokinesis, depletion of PRC1 and KIF4A displayed different phenotypes. Cells depleted of PRC1 failed to form a polarized microtubule array or ectopic furrows following mitotic exit, and recruitment of Aurora B kinase, male germ cell Rac GTPase-activating protein, and RhoA to the cortex was impaired. In contrast, KIF4A depletion impaired neither polarization nor ectopic furrowing, but it did result in elongated spindles with a diffuse distribution of cytokinetic factors. Thus, even in the absence of spindle bipolarity, PRC1 appears to be essential for polarizing parallel microtubules and concentrating the factors responsible for contractile ring assembly, whereas KIF4A is required for limiting the length of anaphase microtubules.     

The molecular mechanism of targeted vesicle transport in cytokinesis

Recent studies have demonstrated that vesicle transport to cleavage furrow is indispensable for cytokinesis. Some animal and plant cells form distinct structures during cell division known as central spindle and phragmoplast, respectively. Several essential factors involved in the vesicle transport have been isolated so far. SNARE proteins and molecular motors play a central role in this process. For future research of cytokinesis, it is important to investigate these factors as well as cytoskeletal components of the contractile ring in detail. This review focuses on the molecular mechanism of targeted vesicle transport in cytokinesis.     

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

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

Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.     

Kinesins to the core: The role of microtubule-based motor proteins in building the mitotic spindle midzone

In mammalian cultured cells the initiation of cytokinesis is regulated – both temporally and spatially – by the overlapping, anti-parallel microtubules of the spindle midzone. This region recruits several key central spindle components: PRC-1, polo-like kinase 1 (Plk-1), the centralspindlin complex, and the chromosome passenger complex (CPC), which together serve to stabilize the microtubule overlap, and also to coordinate the assembly of the cortical actin/myosin cytoskeleton necessary to physically cleave the cell in two. The localization of these crucial elements to the spindle midzone requires members of the kinesin superfamily of microtubule-based motor proteins. Here we focus on reviewing the role played by a variety of kinesins in both building and operating the spindle midzone machinery during cytokinesis.     

Phosphorylation of MyoGEF on Thr-574 by Plk1 promotes MyoGEF localization to the central spindle

We reported previously that a guanine nucleotide exchange factor, MyoGEF, localizes to the central spindle, activates RhoA, and is required for cytokinesis. In this study, we have found that Plk1 (polo-like kinase 1) can phosphorylate MyoGEF, thereby recruiting MyoGEF to the central spindle as well as enhancing MyoGEF activity toward RhoA. The in vitro kinase assay shows that Plk1 can phosphorylate MyoGEF on threonine 574. Immunoprecipitation/immunoblot analysis demonstrates that mutation of threonine 574 to alanine dramatically decreases threonine phosphorylation of MyoGEF in transfected HeLa cells, suggesting that threonine 574 is phosphorylated in vivo. Consistent with these observations, immunofluorescence shows that Plk1 and MyoGEF colocalize at the spindle pole and central spindle during mitosis and cytokinesis. Importantly, RNA interference-mediated depletion of Plk1 interferes with the localization of MyoGEF at the spindle pole and central spindle. Moreover, mutation of threonine 574 to alanine in MyoGEF or depletion of Plk1 by RNA interference leads to a decrease in MyoGEF activity toward RhoA in HeLa cells. Therefore, our results suggest that Plk1 can regulate MyoGEF activity and localization, contributing to the regulation of cytokinesis.     

Cell cycle control of spindle elongation

Different organisms employ a variety of strategies to segregate their chromosomes during mitosis. Despite these differences, however, the basic regulatory principles that govern this intricate process are evolutionarily conserved. Above all, rapid dephosphorylation of mitotic phosphoproteins upon the metaphase-to-anaphase transition has proven to be essential for proper function of the mitotic spindle and accurate chromosome segregation in all eukaryotes. Recently, a central midzone component, the microtubule crosslinker Ase1/PRC1 (anaphase spindle elongation 1/protein regulating cytokinesis 1), was uncovered as a universal target of such control mechanism. Depending on its phosphorylation status, Ase1 either restrains spindle elongation in metaphase or promotes it after anaphase onset via recruitment of kinesin motor proteins to the midzone. Here we discuss the potential role of Ase1/PRC1 as a central regulatory platform that interconnects distinct functions of the midzone such as spindle stability, spindle elongation and cytokinesis. Additionally, we provide a comparative overview of the chromosome segregation strategies used by the main model organisms.     

Modulated targeting of GFP-AtMAP65-1 to central spindle microtubules during division

AtMAP65-1 bundles cortical microtubules and we examined how this property is regulated during division in time-lapse studies of Arabidopsis suspension cells expressing GFP-AtMAP65-1. Spindle fluorescence is diffuse during metaphase, restored to the central spindle at anaphase and then compacted at the midline during late anaphase/early telophase. However, mutagenesis of the microtubule-associated protein (MAP) consensus Cdk site to a non-phosphorylatable form allows premature decoration of microtubules traversing the central region of the metaphase spindle without affecting the timing of the subsequent compaction. This suggests that mutagenesis does not affect compaction but does affect a phosphorylation/dephosphorylation switch that normally targets AtMAP65-1 to the central spindle at the metaphase/anaphase transition. GFP-AtMAP65-1 continues to label the midline of the early phragmoplast, suggesting a structural continuity with the central spindle - both structures being composed of anti-parallel microtubules. However, once the cytokinetic apparatus expands into a ring the MAP becomes depleted at the midline. Despite this, cytokinesis is not arrested and membrane and callose are deposited at the cell plate. It is concluded that AtMAP65-1 plays a role in the central spindle at anaphase to early cytokinesis but is not essential at the midline of the phragmoplast at later stages.     

Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly

During cytokinesis, the organization of the spindle midzone and chromosome segregation is controlled by the central spindle, a microtubule cytoskeleton containing kinesin motors and non‐motor microtubule‐associated proteins. The anaphase spindle elongation 1/protein regulator of cytokinesis 1/microtubule associated protein 65 (Ase1/PRC1/MAP65) family of microtubule‐bundling proteins are key regulators of central spindle assembly, mediating microtubule crosslinking and spindle elongation in the midzone. Ase1/PRC1/MAP65 serves as a complex regulatory platform for the recruitment of other midzone proteins at the spindle midzone. Herein, we summarize recent advances in understanding of the structural domains and molecular kinetics of the Ase1/PRC1/MAP65 family. We summarize the regulatory network involved in post‐translational modifications of Ase1/PRC1 by cyclin‐dependent kinase 1 (Cdk1), cell division cycle 14 (Cdc14) and Polo‐like kinase 1 (Plk1) and also highlight multiple functions of Ase1/PRC1 in central spindle organization, spindle elongation and cytokinesis during cell division.     

Role of Survivin in cytokinesis revealed by a separation-of-function allele

The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. Here we identify a missense allele of Drosophila Survivin that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this "separation-of-function" allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems.