Maternal phosphatase inhibitor-2 is required for proper chromosome segregation and mitotic synchrony during Drosophila embryogenesis

Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase conserved among all eukaryotes, present as the essential GLC7 gene in yeast. Inhibitor-2 (I-2) is an ancient PP1 regulator, named GLC8 in yeast, but its in vivo function is unknown. Unlike mammals with multiple I-2 genes, in Drosophila there is a single I-2 gene, and here we describe its maternally derived expression and required function during embryogenesis. During oogenesis, germline expression of I-2 results in the accumulation of RNA and abundant protein in unfertilized eggs; in embryos, the endogenous I-2 protein concentrates around condensed chromosomes during mitosis and also surrounds interphase nuclei. An I-2 loss-of-function genotype is associated with a maternal-effect phenotype that results in drastically reduced progeny viability, as measured by reduced embryonic hatch rates and larval lethality. Embryos derived from I-2 mutant mothers show faulty chromosome segregation and loss of mitotic synchrony in cleavage-stage embryos, patchy loss of nuclei in syncytial blastoderms, and cuticular pattern defects in late-stage embryos. Transgenic expression of wild-type I-2 in mutant mothers gives dose-dependent rescue of the maternal effect on embryo hatch rate. We propose that I-2 is required for proper chromosome segregation during Drosophila embryogenesis through the coordinated regulation of PP1 and Aurora B.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.     

Aurora B Interaction of Centrosomal Nlp Regulates Cytokinesis

Cytokinesis is a fundamental cellular process, which ensures equal abscission and fosters diploid progenies. Aberrant cytokinesis may result in genomic instability and cell transformation. However, the underlying regulatory machinery of cytokinesis is largely undefined. Here, we demonstrate that Nlp (Ninein-like protein), a recently identified BRCA1-associated centrosomal protein that is required for centrosomes maturation at interphase and spindle formation in mitosis, also contributes to the accomplishment of cytokinesis. Through immunofluorescent analysis, Nlp is found to localize at midbody during cytokinesis. Depletion of endogenous Nlp triggers aborted division and subsequently leads to multinucleated phenotypes. Nlp can be recruited by Aurora B to the midbody apparatus via their physical association at the late stage of mitosis. Disruption of their interaction induces aborted cytokinesis. Importantly, Nlp is characterized as a novel substrate of Aurora B and can be phosphorylated by Aurora B. The specific phosphorylation sites are mapped at Ser-185, Ser-448, and Ser-585. The phosphorylation at Ser-448 and Ser-585 is likely required for Nlp association with Aurora B and localization at midbody. Meanwhile, the phosphorylation at Ser-185 is vital to Nlp protein stability. Disruptions of these phosphorylation sites abolish cytokinesis and lead to chromosomal instability. Collectively, these observations demonstrate that regulation of Nlp by Aurora B is critical for the completion of cytokinesis, providing novel insights into understanding the machinery of cell cycle progression.     

Calcium- and integrin-binding protein 1 regulates microtubule organization and centrosome segregation through polo like kinase 3 during cell cycle progression

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that are involved in the regulation of the various stages of the cell cycle. Plk2 and Plk3, two members of this family, are known to interact with calcium- and integrin-binding protein 1 (CIB1). Activity of both Plk2 and Plk3 is inhibited by CIB1 in a calcium-dependent manner. However, the physiological consequences of this inhibition are not known. Here, we show that overexpression of CIB1 inhibits T47D cell proliferation. Overexpression of CIB1 or knockdown of Plk3 using shRNA produced a multinucleated phenotype in T47D cells. This phenotype was not cancer cell specific, since it also occurred in normal cells. The cells overexpressing CIB1 appear to undergo proper nuclear division, but are unable to complete the process of cytokinesis, thus forming large multinucleated cells. Both CIB1 overexpression and Plk3 knockdown disrupted microtubule organization and centrosomal segregation, which may have led to incomplete cytokinesis. The observed effect of CIB1 overexpression is not due to the inhibition of Plk2 by CIB1. Plk3 and CIB1 both colocalize at the centrosomes, however, localization of CIB1 is dependent on the expression of Plk3. Furthermore, expression of Plk3 blocks the multinucleated phenotype induced by expression of CIB1 in these cells. These results suggest that CIB1 tightly regulates Plk3 activity during cell division and that either over- or underexpression results in a multinucleated phenotype.     

Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number

The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over- and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during cytokinesis.     

Phosphoregulation of MgcRacGAP in mitosis involves Aurora B and Cdk1 protein kinases and the PP2A phosphatase

MgcRacGAP, a Rho GAP essential to cytokinesis, works both as a Rho GTPase regulator and as a scaffolding protein. MgcRacGAP interacts with MKLP1 to form the centralspindlin complex and associates with the RhoGEF Ect2. The GAP activity of MgcRacGAP is regulated by Aurora B phosphorylation. We have isolated B56epsilon, a PP2A regulatory subunit, as a new MgcRacGAP partner. We report here that (i) MgcRacGAP is phosphorylated by Aurora B and Cdk1, (ii) PP2A dephosphorylates Aurora B and Cdk1 phosphorylated sites and (iii) inhibition of PP2A abrogates MgcRacGAP/Ect2 interaction. Therefore, PP2A may regulate cytokinesis by dephosphorylating MgcRacGAP and its interacting partners.     

Phosphorylation and functions of inhibitor-2 family of proteins

Protein phosphatase-1 (PP1) is an essential protein Ser/Thr phosphatase that is extraordinarily conserved from yeast to human, and Inhibitor-2 (I-2) is the most ancient of the heat-stable proteins specific for PP1. We identified novel I-2 homologues in Caenorhabditis elegans (Ce) and Xenopus laevis (Xe) and compared them to the I-2 proteins from Homo sapiens (Hs), Saccharomyces cerevisiae (GLC8), and Drosophila melanogaster (Dm). The Ce I-2 and Dm I-2 showed the highest potency inhibition of rabbit PP1 with IC50 near 5 nM compared to Hs I-2 and Xe I-2 with IC50 between 10 and 50 nM and GLC8 with >100-fold lower activity. Inhibition of PP1 bound to Nek2 kinase activated the kinase to phosphorylate a C-Nap1 domain substrate. All the species of I-2 except GLC8 activated the Nek2::PP1 to the same extent as microcystin-LR. Only Hs I-2 and Xe I-2, not the I-2 proteins more divergent in sequence, directly activated human Aurora-A kinase. Various species of I-2 have a common PxTP phosphorylation site that showed a wide range of reactivity with GSK3, ERK, or CDC2/cyclinB1 kinases. The Suc1 subunit of CDC2/cyclinB1 enhanced reactivity with I-2, consistent with this being a site of mitotic phosphorylation. The results show species specificity among the I-2 family within the context of conserved PP1 inhibitory activity and variable phosphorylation by Pro-directed kinases.     

The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation

Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC), consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and cytokinetic initiation. This is the first time to our knowledge that the dual functions of an Aurora B homolog is dissected and separated into two clearly distinct time frames in a cell cycle.     

Targeting the INCENP IN-box–Aurora B interaction to inhibit CPC activity in vivo

The chromosome passenger complex (CPC) is an essential regulator of mitosis and cytokinesis. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), and the targeting subunits survivin and borealin/Dasra B. INCENP is a scaffolding subunit for the CPC and activates Aurora B via its conserved IN-box domain. We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function. The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo. We then screened a targeted library of small (five to nine residues long) circular peptide (CP) IN-box fragments generated using split intein circular ligation of proteins and peptides (SICLOPPS) methodology. We identified a number of CPs that caused modest but reproducible increases in rates of multinucleated and micronucleated cells. Our results provide proof of concept that inhibition of the Aurora B–IN-box interaction is a viable strategy for interfering with CPC function in vivo.     

The novel function of JADE1S in cytokinesis of epithelial cells

JADE1 belongs to a small family of PHD zinc finger proteins that interacts with histone acetyl transferase (HAT) HBO1 and is associated with chromatin. We recently reported JADE1 chromatin shuttling and phosphorylation during G2/M to G1 transition, which was sensitive to Aurora A inhibition. In the current study we examined mechanisms of the cell cycle regulation by the small isoform of JADE1 protein, JADE1S, and report data showing that JADE1S has a novel function in the regulation of cytokinesis. Using FACS assays, we show that, JADE1S depletion facilitated rates of G1-cells accumulation in synchronously dividing HeLa cell cultures. Depletion of JADE1S protein in asynchronously dividing cells decreased the proportion of cytokinetic cells, and increased the proportion of multi-nuclear cells, indicative of premature and failed cytokinesis. In contrast, moderate overexpression of JADE1S increased the number of cytokinetic cells in time- and dose- dependent manner, indicating cytokinetic delay. Pharmacological inhibition of Aurora B kinase resulted in the release of JADE1S-mediated cytokinetic delay and allowed progression of abscission in cells over-expressing JADE1S. Finally, we show that JADE1S protein localized to centrosomes in interphase and mitotic cells, while during cytokinesis JADE1S localized to the midbody. Neither JADE1L nor partner of JADE1, HAT HBO1 was localized to the centrosomes or midbodies. Our study identifies the novel role for JADE1S in regulation of cytokinesis and suggests function in Aurora B kinase-mediated cytokinesis checkpoint.     

CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.     

The cell cycle as a therapeutic target against Trypanosoma brucei: Hesperadin inhibits Aurora kinase-1 and blocks mitotic progression in bloodstream forms

Aurora kinase family members co-ordinate a range of events associated with mitosis and cytokinesis. Anti-cancer therapies are currently being developed against them. Here, we evaluate whether Aurora kinase-1 (TbAUK1) from pathogenic Trypanosoma brucei might be targeted in anti-parasitic therapies as well. Conditional knockdown of TbAUK1 within infected mice demonstrated its essential contribution to infection. An in vitro kinase assay was developed which used recombinant trypanosome histone H3 as a substrate. Tandem mass spectroscopy identified a novel phosphorylation site in the carboxyl-tail of recombinant trypanosome histone H3. Hesperadin, an inhibitor of human Aurora B, prevented the phosphorylation of substrate with IC₅₀ of 40 nM. Growth of cultured bloodstream forms was also sensitive to Hesperadin (IC₅₀ of 50 nM). Hesperadin blocked nuclear division and cytokinesis but not other aspects of the cell cycle. Consequently, growth arrested cells accumulated multiple kinetoplasts, flagella and nucleoli, similar to the effects of RNAi-dependent knockdown of TbAUK1 in cultured bloodstream forms cells. Molecular models predicted high-affinity binding of Hesperadin to both conserved and novel sites in TbAUK1. Collectively, these data demonstrate that cell cycle progression is essential for infections with T. brucei and that parasite Aurora kinases can be targeted with small-molecule inhibitors.     

Dynamic Phosphorylation of NudC by Aurora B in Cytokinesis

Nuclear distribution protein C (NudC) is a mitotic regulator that plays a role in cytokinesis. However, how NudC is regulated during cytokinesis remains unclear. Here, we show that NudC is phosphorylated by Aurora B, a kinase critical for cell abscission. NudC is co-localized with Aurora B at the midbody and co-immunoprecipitated with Aurora B in mitosis. Inhibition of Aurora B by ZM447439 reduced NudC phosphorylation, suggesting that NudC is an Aurora B substrate in vivo. We identified T40 on NudC as an Aurora B phosphorylation site. NudC depletion resulted in cytokinesis failure with a dramatic elongation of the intercellular bridge between daughter cells, sustained Aurora B activity at the midbody, and reduced cell abscission. These cytokinetic defects can be rescued by the ectopic expression of wild-type NudC. Reconstitution with T40A phospho-defective NudC was found to rescue the cytokinesis defect. In contrast, reconstitution with the T40D phospho-mimetic NudC was inefficient in supporting the completion of cytokinesis. These results suggest that that dynamic phosphorylation of NudC by Aurora B regulates cytokinesis.     

The Chromosomal Passenger Complex and a Mitotic Kinesin Interact with the Tousled-Like Kinase in Trypanosomes to Regulate Mitosis and Cytokinesis

Aurora B kinase plays essential roles in mitosis and cytokinesis in eukaryotes. In the procyclic form of Trypanosoma brucei, the Aurora B homolog TbAUK1 regulates mitosis and cytokinesis, phosphorylates the Tousled-like kinase TbTLK1, interacts with two mitotic kinesins TbKIN-A and TbKIN-B and forms a novel chromosomal passenger complex (CPC) with two novel proteins TbCPC1 and TbCPC2. Here we show with time-lapse video microscopy the time course of CPC trans-localization from the spindle midzone in late anaphase to the dorsal side of the cell where the anterior end of daughter cell is tethered, and followed by a glide toward the posterior end to divide the cell, representing a novel mode of cytokinesis in eukaryotes. The three subunits of CPC, TbKIN-B and TbTLK1 interact with one another suggesting a close association among the five proteins. An ablation of TbTLK1 inhibited the subsequent trans-localization of CPC and TbKIN-B, whereas a knockdown of CPC or TbKIN-B disrupted the spindle pole localization of TbTLK1 during mitosis. In the bloodstream form of T. brucei, the five proteins also play essential roles in chromosome segregation and cytokinesis and display subcellular localization patterns similar to that in the procyclic form. The CPC in bloodstream form also undergoes a trans-localization during cytokinesis similar to that in the procyclic form. All together, our results indicate that the five-protein complex CPC-TbTLK1-TbKIN-B plays key roles in regulating chromosome segregation in the early phase of mitosis and that the highly unusual mode of cytokinesis mediated by CPC occurs in both forms of trypanosomes.     

Late mitotic functions of Aurora kinases

The coordination between late mitotic events such as poleward chromosome motion, spindle elongation, DNA decondensation, and nuclear envelope reformation (NER) is crucial for the completion of chromosome segregation at the anaphase-telophase transition. Mitotic exit is driven by a decrease of Cdk1 kinase activity and an increase of PP1/PP2A phosphatase activities. More recently, Aurora kinases have also emerged as master regulators of late mitotic events and cytokinesis. Aurora A is mainly associated with spindle poles throughout mitosis and midbody during telophase, whereas Aurora B re-localizes from centromeres in early mitosis to the spindle midzone and midbody as cells progress from anaphase to the completion of cytokinesis. Functional studies, together with the identification of a phosphorylation gradient during anaphase, established Aurora B as a major player in the organization of the spindle midzone and in the spatiotemporal coordination between chromosome segregation and NER. Aurora A has been less explored, but a cooperative role in spindle midzone stability has also been proposed, implying that both Aurora A and B contribute to accurate chromosome segregation during mitotic exit. Here, we review the roles of the Aurora kinases in the regulation of late mitotic events and discuss how they work together with other mitotic players to ensure an error-free mitosis.     

KIF4A and PP2A–B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central spindle

The mitotic kinase Aurora B is concentrated at the anaphase central spindle by the kinesin MKlp2 during mitotic exit and cytokinesis. This pool of Aurora B phosphorylates substrates including the kinesin KIF4A to regulate central spindle length. In this paper, we identify a counteracting system in which PP2A–B56γ and -ε, but not PP2A–B56α, -β, and -δ, are maintained at the central spindle by KIF4A. Biochemical assays show that PP2A–B56γ can dephosphorylate the T799 Aurora B site on KIF4A and thereby counteract the Aurora B– and microtubule-stimulated ATPase activity of KIF4A. In agreement with these observations, combined silencing of PP2A–B56γ and -ε resulted in increased phosphorylation of KIF4A T799 and decreased central spindle growth in anaphase B. Furthermore, reduced turnover of regulatory phosphorylation on another Aurora B substrate MKlp1 was observed, suggesting that PP2A–B56γ and -ε play a general role opposing Aurora B at the central spindle. KIF4A and PP2A–B56γ and -ε therefore create a spatially restricted negative feedback loop counteracting Aurora B in anaphase.     

Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells.