p27Kip1 regulates the microtubule bundling activity of PRC1.

Cytokinesis begins in anaphase with the formation of the central spindle. PRC1 is a microtubule associated protein that plays an essential role in central spindle formation by crosslinking antiparallel microtubules. We have identified PRC1 as a novel binding partner for p27Kip1 (p27). p27 is a cyclin-CDK inhibitor that causes cell cycle arrest in G1. However, p27 has also been involved in the regulation of G2/M progression and cytokinesis, as well as of other cellular processes, including actin and microtubule cytoskeleton dynamics. We found that p27 interferes with the ability of PRC1 to bind to microtubules, without affecting PRC1 dimerization or its capacity to interact with other partners such as KIF4. In this way, p27 inhibited microtubule bundling by PRC1 in vitro and prevented the extensive microtubule bundling phenotype caused by PRC1 overexpression in cells in culture. Finally, co-expression of p27 or a p27 mutant that does not bind cyclin-CDKs inhibited multinucleation induced by PRC1 overexpression. Together, our results suggest that p27 may participate in the regulation of mitotic progression in a CDK-independent manner by modulating PRC1 activity.

WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.

We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.

Failure of cell cleavage induces senescence in tetraploid primary cells.

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-ß-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

CENP-A is essential for cardiac progenitor cell proliferation.

Centromere protein A (CENP-A) is a homolog of histone H3 that epigenetically marks the heterochromatin of chromosomes. CENP-A is a critical component of the cell cycle machinery that is necessary for proper assembly of the mitotic spindle. However, the role of CENP-A in the heart and cardiac progenitor cells (CPCs) has not been previously studied. This study shows that CENP-A is expressed in CPCs and declines with age. Silencing CENP-A results in a decreased CPC growth rate, reduced cell number in phase G 2/M of the cell cycle, and increased senescence associated ß-galactosidase activity. Lineage commitment is not affected by CENP-A silencing, suggesting that cell cycle arrest induced by loss of CENP-A is a consequence of senescence and not differentiation. CENP-A knockdown does not exacerbate cell death in undifferentiated CPCs, but increases apoptosis upon lineage commitment. Taken together, these results indicate that CPCs maintain relatively high levels of CENP-A early in life, which is necessary for sustaining proliferation, inhibiting senescence, and promoting survival following differentiation of CPCs.

TD-60 is required for interphase cell cycle progression.

We previously identified TD-60 (RCC2) as a mitotic centromere-associated protein that is necessary for proper completion of mitosis. We now report that TD-60 is an essential regulator of cell cycle progression during interphase. siRNA suppression blocks progression of mammalian G1/S phase cells and progression of G2 cells into mitosis. Prolonged arrest occurs both in non-transformed cells and in transformed cells lacking functional p53. TD-60 associates with Rac1 and Arf6 and has recently been demonstrated to be an element of α5ß1 integrin and cortactin interactomes. These associations with known elements of cell cycle control, together with our data, suggest that TD-60 is an essential component of one or more signaling pathways that drive cell cycle progression. During mitosis, TD-60 is required for correct assembly of the mitotic spindle and activation of key mitotic proteins. In contrast, in interphase TD-60 promotes cell cycle progression through what must be distinct mechanisms. TD-60 thus appears to be one of the growing categories of proteins that "moonlight," or have more than one distinct cellular function.

Glioblastoma motility occurs in the absence of actin polymer.

In fibroblasts and keratocytes, motility is actin dependent, while microtubules play a secondary role, providing directional guidance. We demonstrate here that the motility of glioblastoma cells is exceptional, in that it occurs in cells depleted of assembled actin. Cells display persistent motility in the presence of actin inhibitors at concentrations sufficient to fully disassemble actin. Such actin independent motility is characterized by the extension of cell protrusions containing abundant microtubule polymers. Strikingly, glioblastoma cells exhibit no motility in the presence of microtubule inhibitors, at concentrations that disassemble labile microtubule polymers. In accord with an unconventional mode of motility, glioblastoma cells have some unusual requirements for the Rho GTPases. While Rac1 is required for lamellipodial protrusions in fibroblasts, expression of dominant negative Rac1 does not suppress glioblastoma migration. Other GTPase mutants are largely without unique effect, except dominant positive Rac1-Q61L, and rapidly cycling Rac1-F28L, which substantially suppress glioblastoma motility. We conclude that glioblastoma cells display an unprecedented mode of intrinsic motility that can occur in the absence of actin polymer, and that appears to require polymerized microtubules.

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage.

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.

SMC5 and MMS21 are required for chromosome cohesion and mitotic progression.

Members of the structural maintenance of chromosome (SMC) protein family have essential functions during mitosis, ensuring chromosome condensation (SMC2/4) and cohesion (SMC1/3). The SMC5/6 complex has been implicated in a variety of DNA maintenance processes but unlike the other SMC proteins, SMC5/6 have not been attributed any role in mitosis. Here, we find that ablation of either SMC5 or the SUMO-ligase MMS21 leads to premature sister chromatid separation prior to anaphase. The failure of normal chromosome alignment activates the spindle assembly checkpoint and blocks mitotic progression. Interestingly, there is no similar mitotic response to ablation of SMC6. Further, we show that mitotic SMC5 co-elutes from column fractions that contain MMS21 but lack SMC6. Our results thus establish that SMC5 is crucial for mitotic progression and maintenance of sister chromatid cohesion during mitosis, and that this role of SMC5 seems to be independent of the SMC5/6 complex.

Molecular distinctions between Aurora A and B: a single residue change transforms Aurora A into correctly localized and functional Aurora B.

Aurora A and Aurora B, paralogue mitotic kinases, share highly similar primary sequence. Both are important to mitotic progression, but their localizations and functions are distinct. We have combined shRNA suppression with overexpression of Aurora mutants to address the cause of the distinction between Aurora A and Aurora B. Aurora A residue glycine 198 (G198), mutated to asparagine to mimic the aligned asparagine 142 (N142) of Aurora B, causes Aurora A to bind the Aurora B binding partner INCENP but not the Aurora A binding partner TPX2. The mutant Aurora A rescues Aurora B mitotic function. We conclude that binding to INCENP is alone critical to the distinct function of Aurora B. Although G198 of Aurora A is required for TPX2 binding, N142G Aurora B retains INCENP binding and Aurora B function. Thus, although a single residue change transforms Aurora A, the reciprocal mutation of Aurora B does not create Aurora A function. An Aurora A-Delta120 N-terminal truncation construct reinforces Aurora A similarity to Aurora B, because it does not associate with centrosomes but instead associates with kinetochores.

Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed.

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)-dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.

Tetraploidy and tumor development.

In tumorigenesis, aneuploidy is frequently preceded by tetraploidy. Major issues include how tetraploidy arises and how cells can effectively respond to this state. Two recent papers address these issues. Shi and King demonstrate that nondisjunction of chromosomes in mitosis frequently results in tetraploidy through mitotic cleavage failure. Fujiwara et al. demonstrate that p53 null tetraploid cells are highly competent to induce tumors in nude mice. Together, these papers emphasize the unique hazard of tetraploidy and the fact that p53 status has an intrinsic capacity to eliminate tetraploid cells and suppress tumorigenesis. This p53-dependent elimination may represent a checkpoint control.

A small C-terminal sequence of Aurora B is responsible for localization and function.

Aurora B, a protein kinase required in mitosis, localizes to inner centromeres at metaphase and the spindle midzone in anaphase and is required for proper chromosome segregation and cytokinesis. Aurora A, a paralogue of Aurora B, localizes instead to centrosomes and spindle microtubules. Except for distinct N termini, Aurora B and Aurora A have highly similar sequences. We have combined small interfering RNA (siRNA) ablation of Aurora B with overexpression of truncation mutants to investigate the role of Aurora B sequence in its function. Reintroduction of Aurora B during siRNA treatment restored its localization and function. This permitted a restoration of function test to determine the sequence requirements for Aurora B targeting and function. Using this rescue protocol, neither N-terminal truncation of Aurora B unique sequence nor substitution with Aurora A N-terminal sequence affected Aurora B localization or function. Truncation of unique Aurora B C-terminal sequence from terminal residue 344 to residue 333 was without effect, but truncation to 326 abolished localization and function. Deletion of residues 326-333 completely abolished localization and blocked cells at prometaphase, establishing this sequence as critical to Aurora B function. Our findings thus establish a small sequence as essential for the distinct localization and function of Aurora B.

Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not.

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

Bub1, a gatekeeper for Cdc20-dependent mitotic exit.

The mitotic spindle assembly checkpoint arrests cells at metaphase by suppressing Cdc20, a protein required to trigger ubiquitination and consequent degradation of cyclin B. New evidence from Tang et al. appearing in the November 5th issue of Molecular Cell finds that one of the checkpoint proteins, Bub1, specifically phosphorylates Cdc20 to suppress APC/C activation.

Distinct dynamics of Aurora B and Survivin during mitosis.

We have studied the dynamics of Aurora B and Survivin during mitosis in living cells, using C-terminal GFP chimeras of the two proteins. These chimeras showed identical localization and behave as bona fide wild type proteins. The mobility of Aurora B-GFP and Survivin-GFP was analyzed by FRAP. The data show that Survivin-GFP, in contrast to Aurora B-GFP, is highly mobile at prometaphase and metaphase. At telophase and cell cleavage, both chimeras are found to be fully immobile. The ablation of Aurora B by siRNA results in a dramatic decrease of the Survivin-GFP mobility. These results demonstrate that Survivin, but not Aurora B, is weakly associated with the centromeric chromatin at prometaphase and metaphase. The weak association of Survivin with centromeric chromatin is dependent on the presence of Aurora B and is not affected by treatment with either nocodazole or taxol. The rapid and conditional interchange between passenger proteins that we show by live imaging indicates that the high affinity interactions demonstrated with in vitro analysis of passenger protein binding are, in fact, static "snapshots" of highly dynamic and regulated in vivo interactions in mitotic cells.

In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities.

Human Eg5, a member of the kinesin superfamily, plays a key role in mitosis, as it is required for the formation of a bipolar spindle. We describe here the first in vitro microtubule-activated ATPase-based assay for the identification of small-molecule inhibitors of Eg5. We screened preselected libraries obtained from the National Cancer Institute and identified S-trityl-L-cysteine as the most effective Eg5 inhibitor with an IC50 of 1.0 micromol/L for the inhibition of basal ATPase activity and 140 nmol/L for the microtubule-activated ATPase activity. Subsequent cell-based assays revealed that S-trityl-L-cysteine induced mitotic arrest in HeLa cells (IC50, 700 nmol/L) with characteristic monoastral spindles. S-trityl-L-cysteine is 36 times more potent for inducing mitotic arrest than the well-studied inhibitor, monastrol. Gossypol, flexeril, and two phenothiazine analogues were also identified as Eg5 inhibitors, and we found that they all result in monoastral spindles in HeLa cells. It is notable that all the Eg5 inhibitors identified here have been shown previously to inhibit tumor cell line growth in the NCI 60 tumor cell line screen, and we conclude that their antitumor activity may at least in part be explained by their ability to inhibit Eg5 activity.

Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase.

DNA damage by double-strand breaks induces arrest during interphase in mammalian cells. It is not clear whether DNA damage can arrest cells in mitosis. We show here that three human cell lines, HeLa, U2OS, and HCT116, do not delay in mitosis in response to double-strand breaks induced during mitosis by gamma irradiation or by adriamycin. Durable arrest at metaphase occurs, however, with ICRF-193, a topoisomerase II inhibitor that does not damage DNA. Arrest with ICRF-193 is not accompanied by recruitment of Mad2 or Bub1 to kinetochores, nor by phosphorylation of the histone H2AX, indicating arrest by ICRF-193 is not due to activation of the spindle assembly checkpoint, nor is it a response to DNA damage. VP-16, another decatenation inhibitor, induces metaphase arrest only at concentrations well above those that induce DNA damage. We conclude that decatenation failure, but not DNA damage, creates metaphase arrest in mammalian cells.