The FEAR Before MEN: networks of mitotic exit

Variations in the normal regulation of the mitotic cell cycle can lead to such global cellular changes as differential development or malignant transformation. Studies on the control of mitosis are particularly important to discover the details of the basic mechanisms responsible for normal cell division, as well as to learn about strategies employed by cancerous cells to indefinitely proliferate. The past years have brought noteworthy progress in elucidating the molecular pathways that regulate crucial events during mitosis such as: chromosome condensation, formation of the mitotic spindle, chromosome segregation, cytokinesis, and disassembly of the mitotic spindle.     

Hec1 Contributes to Mitotic Centrosomal Microtubule Growth for Proper Spindle Assembly through Interaction with Hice1

Previous studies have stipulated Hec1 as a conserved kinetochore component critical for mitotic control in part by directly binding to kinetochore fibers of the mitotic spindle and by recruiting spindle assembly checkpoint proteins Mad1 and Mad2. Hec1 has also been reported to localize to centrosomes, but its function there has yet to be elucidated. Here, we show that Hec1 specifically colocalizes with Hice1, a previously characterized centrosomal microtubule-binding protein, at the spindle pole region during mitosis. In addition, the C-terminal region of Hec1 directly binds to the coiled-coil domain 1 of Hice1. Depletion of Hice1 by small interfering RNA (siRNA) reduced levels of Hec1 in the cell, preferentially at centrosomes and spindle pole vicinity. Reduction of de novo microtubule nucleation from mitotic centrosomes can be observed in cells treated with Hec1 or Hice1 siRNA. Consistently, neutralization of Hec1 or Hice1 by specific antibodies impaired microtubule aster formation from purified mitotic centrosomes in vitro. Last, disruption of the Hec1/Hice1 interaction by overexpressing Hice1ΔCoil1, a mutant defective in Hec1 interaction, elicited abnormal spindle morphology often detected in Hec1 and Hice1 deficient cells. Together, the results suggest that Hec1, through cooperation with Hice1, contributes to centrosome-directed microtubule growth to facilitate establishing a proper mitotic spindle.     

LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 in hibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.     

The FEAR Before MEN: Networks of Mitotic Exit

Variations in the normal regulation of the mitotic cell cycle can lead to such global cellular changes as differential development or malignant transformation. Studies on the control of mitosis are particularly important to discover the details of the basic mechanisms responsible for normal cell division, as well as to learn about strategies employed by cancerous cells to indefinitely proliferate. The past years have brought noteworthy progress in elucidating the molecular pathways that regulate crucial events during mitosis such as: chromosome condensation, formation of the mitotic spindle, chromosome segregation, cytokinesis, and disassembly of the mitotic spindle.

Key Words:

Mitosis, FEAR, MEN     

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

Overexpression of cyclin A in human HeLa cells induces detachment of kinetochores and spindle pole/centrosome overproduction

The combination of hydroxyurea (HU) and caffeine has been used for inducing kinetochore dissociation from mitotic chromosomes and for causing centrosome/spindle pole amplification. However, these effects on microtubule organizing centers (MTOCs) are limited to certain cell types. It was reasoned that if the biochemical differences in MTOC behavior between cells following HU treatment could be identified, then critical information concerning the regulation of these organelles would be obtained. During these studies, it was determined that cells from hamster, rat, and deer could be induced to enter mitosis with dissociated kinetochores and to synthesize centrosomes during arrest with HU, while cells from human and mouse could not. Comparisons between human HeLa cells and CHO cells determined that cyclin A levels were depressed in HeLa cells relative to CHO cells following HU addition. Overexpression of cyclin A in HeLa cells converted them to a cell type capable of detaching kinetochores from mitotic chromosomes. Ultrastructural analyses determined that the detached human kinetochores exhibited a normal plate-like morphology and appeared capable of associating with microtubules. In addition, HeLa cells overexpressing cyclin A also overproduced spindle poles during HU arrest, demonstrating that cyclin A activity also is important for centrosome replication during interphase. In summary, elevated cyclin A levels are important for the capacity of cells to be driven into mitosis by caffeine addition, for the ability of cells to progress to mitosis with detached kinetochores, and for centrosome/spindle pole replication.     

The mitotic spindle checkpoint is a critical determinant for topoisomerase-based chemotherapy

A novel strategy in cancer therapy is the induction of mitotic cell death by the pharmacological abrogation of cell cycle checkpoints. UCN-01 is such a compound that overrides the G2 cell cycle arrest induced by DNA damage and forces cells into a deleterious mitosis. The molecular pathways leading to mitotic cell death are largely unknown although recent evidence indicates that mitotic cell death represents a special case of apoptosis. Here, we demonstrate that the mitotic spindle checkpoint is activated upon chemotherapeutic treatment with topoisomerase II poisons and UCN-01. Cells that are forced to enter mitosis in the presence of topoisomerase inhibition arrest transiently in a prometaphase like state. By using a novel pharmacological inhibitor of the spindle checkpoint and spindle checkpoint-deficient cells we show that the spindle checkpoint function is required for the mitotic arrest and, most importantly, for efficient induction of mitotic cell death. Thus, our results demonstrate that the mitotic spindle checkpoint is an important determinant for the outcome of a chemotherapy based on the induction of mitotic cell death. Its frequent inactivation in human cancer might contribute to the observed resistance of tumor cells to these chemotherapeutic drugs.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

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.     

Caffeine promotes apoptosis in mitotic spindle checkpoint-arrested cells

The spindle assembly checkpoint arrests cells in mitosis when defects in mitotic spindle assembly or partitioning of the replicated genome are detected. This checkpoint blocks exit from mitosis until the defect is rectified or the cell initiates apoptosis. In this study we have used caffeine to identify components of the mechanism that signals apoptosis in mitotic checkpoint-arrested cells. Addition of caffeine to spindle checkpoint-arrested cells induced >40% apoptosis within 5 h. It also caused proteasome-mediated destruction of cyclin B1, a corresponding reduction in cyclin B1/cdk1 activity, and reduction in MPM-2 reactivity. However, cells retained MAD2 staining at the kinetochores, an indication of continued spindle checkpoint function. Blocking proteasome activity did not block apoptosis, but continued spindle checkpoint function was essential for apoptosis. After systematically eliminating all known targets, we have identified p21-activated kinase PAK1, which has an anti-apoptotic function in spindle checkpoint-arrested cells, as a target for caffeine inhibition. Knockdown of PAK1 also increased apoptosis in spindle checkpoint-arrested cells. This study demonstrates that the spindle checkpoint not only regulates mitotic exit but apoptosis in mitosis through the activity of PAK1.     

The astrin-kinastrin/SKAP complex localizes to microtubule plus ends and facilitates chromosome alignment

Astrin is a mitotic spindle-associated protein required for the correct alignment of all chromosomes at the metaphase plate. Astrin depletion delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset. Here we describe an astrin complex containing kinastrin/SKAP, a novel kinetochore and mitotic spindle protein, and three minor interaction partners: dynein light chain, Plk1, and Sgo2. Kinastrin is the major astrin-interacting protein in mitotic cells, and is required for astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Cells overexpressing or depleted of kinastrin mislocalize astrin and show the same mitotic defects as astrin-depleted cells. Importantly, astrin fails to localize to and track microtubule plus ends in cells depleted of or overexpressing kinastrin. These findings suggest that microtubule plus end targeting of astrin is required for normal spindle architecture and chromosome alignment, and that perturbations of this pathway result in delayed mitosis and nonphysiological separase activation.     

REGγ proteasome activator is involved in the maintenance of chromosomal stability

REGγ is a member of the 11S regulatory particle that activates the 20S proteasome. Studies in REGγ deficient mice indicated an additional role for this protein in cell cycle regulation and proliferation control. In this paper we demonstrate that REGγ protein is equally expressed throughout the cell cycle, but undergoes a distinctive subcellular localization at mitosis. Thus, while in interphase cells REGγ is nuclear, in telophase cells it localizes on chromosomes, suggesting a role in mitotic progression. Furthermore, we found that REGγ overexpression weakens the mitotic arrest induced by spindle damage, allowing premature exit from mitosis, whereas REGγ depletion has the opposite effect, thus reflecting a new REGγ function, unrelated to its role as proteasome activator. Additionally, we found that primary cells from REGγ-/- mice and human fibroblasts with depleted expression of REGγ or overexpressing a dominant negative mutant unable to activate the 20S proteasome, demonstrated a marked aneuploidy (chromosomal gains and losses), supernumerary centrosomes and multipolar spindles. These findings thus underscore a previously uncharacterized function of REGγ in centrosome and chromosomal stability maintenance.     

A novel role for progesterone and progesterone receptor membrane component 1 in regulating spindle microtubule stability during rat and human ovarian cell mitosis

The present studies were designed to assess the roles of progesterone (P4) and Progesterone Receptor Membrane Component 1 (PGRMC1) in regulating mitosis of spontaneously immortalized granulosa cells (SIGCs) and ovarian cancer cells, SKOV-3 cells. Because PGRMC1 has been detected among the proteins of the human mitotic spindle, we theorized that P4 and PGRMC1 could affect mitosis through a microtubule-dependent process. The present study confirms that SIGC growth is slowed by either P4 treatment or transfection of a PGRMC1 antibody. In both cases, slower cell proliferation was accompanied by an increased percentage of mitotic cells, which is consistent with a P4-induced prolongation of the M phase of the cell cycle. In addition, P4 increased the stability of the spindle microtubules, as assessed by the rate of beta-tubulin disassembly in response to cooling. Also, P4 increased spindle microtubule stability of SKOV-3 cells. This effect was mimicked by the depletion of PGRMC1 in these cells. Importantly, P4 did not increase the stability of the microtubules over that observed in PGRMC1-depleted SKOV-3 cells. Immunofluorescent analysis revealed that PGRMC1 is distributed to the spindle apparatus as well as to the centrosomes at metaphase. Further in situ proximity ligation assay revealed that PGRMC1 interacted with beta-tubulin. Taken together, these results suggest that P4 inhibits mitosis of ovarian cells by increasing the stability of the mitotic spindle. Moreover, P4's actions appear to be dependent on PGRMC1's function within the mitotic spindle.     

Requirement of a functional spindle checkpoint for arsenite-induced apoptosis

To understand the potential influence of spindle checkpoint function in response to arsenic trioxide (ATO)-induced apoptosis observed in cancer cell lines, we examined the correlation between activation of the spindle checkpoint and susceptibility to ATO-induced apoptosis in 10 cancer cell lines lacking functional p53. The ability to functionally activate the spindle checkpoint in each cancer cell line was assessed by the induction of mitotic arrest after Taxol treatment. Bromodeoxyuridine (BrdU) pulse-chase analysis of Taxol-treated cell lines with low mitotic arrest showed that they were not arrested at mitosis but divided abnormally, confirming that spindle checkpoint activation was impaired in these cell lines. Our results demonstrate that apoptosis was significantly induced by ATO in cancer cell lines with functional activation of the spindle checkpoint and substantial induction of mitotic arrest. Cell lines with negligible mitotic arrest exhibited little ATO-induced apoptosis. However, no such correlation was observed following treatment of cells with camptothecin, a topoisomerase I inhibitor. Furthermore, attenuation of the spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 and Mad2 in cancer cells that were susceptible to ATO-induced mitotic arrest and apoptosis greatly reduced the induction of mitotic arrest and apoptosis by ATO and increased the formation of micronuclei or multinuclei in survived cells. The marked correlation between ATO-induced mitotic arrest and apoptosis indicates that the induction of apoptosis by ATO was highly dependent on the functional activation of the spindle checkpoint in cancer cells lacking normal p53 function.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

The C-terminus of PARK2 is required for its self-interaction, solubility and role in the spindle assembly checkpoint

PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.     

The C-terminus of PARK2 is required for its self-interaction, solubility and role in the spindle assembly checkpoint

PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.     

The inhibition of Aurora A abrogates the mitotic delay induced by microtubule perturbing agents

The spindle assembly checkpoint functions during mitosis to ensure that chromosomes are properly aligned in mitotic cells prior to the onset of anaphase, thereby ensuring an equal segregation of genetic material to each daughter cell. Defects in the function of this checkpoint lead to aneuploidy, and eventually to cell death or senescence. The Aurora-related kinases, and in particular Aurora B, have been shown to play a role in regulating the spindle assembly checkpoint. In this study, we demonstrate that Aurora A activity is required for maintainance of the spindle assembly checkpoint mediated-mitotic delay induced by microtubule perturbing agents. Inhibition of Aurora A using MLN8054, a selective small-molecule inhibitor of Aurora A, in paclitaxel- or nocodazole-treated cells induces cells to become multinucleated. Using time-lapse microscopy, we demonstrate that the multinucleation phenotype arises via mitotic slippage, which is significantly accelerated upon Aurora A inhibition. Under these conditions, the spindle assembly checkpoint protein BubR1 remains localized to kinetochores prior to mitotic slippage. Moreover, we demonstrate that Aurora B remains active in these mitotic cells, indicating that the mitotic slippage induced by MLN8054 is most likely due to the inhibition of Aurora A. This finding was corroborated by demonstrating that Aurora A depletion using RNA interference in paclitaxel-treated cells also induces multinucleation. Taken together, these results suggest that Aurora A is necessary for the maintenance of the mitotic delay induced in response to microtubule-perturbing agents.     

Positioning and elongation of the fission yeast spindle by microtubule-based pushing

In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.     

FAT10 plays a role in the regulation of chromosomal stability

Aneuploidy is a key process in tumorigenesis. Dysfunction of the mitotic spindle checkpoint proteins has been implicated as a cause of aneuploidy in cells. We have previously reported that FAT10, a member of the ubiquitin-like modifier family of proteins, is overexpressed in several gastrointestinal and gynecological cancers. Here we show that FAT10 interacts with MAD2, a spindle checkpoint protein, during mitosis. Notably, we show that localization of MAD2 at the kinetochore during the prometaphase stage of the cell cycle was greatly reduced in FAT10-overexpressing cells. Furthermore, compared with parental HCT116 cells, fewer mitotic cells were observed after double thymidine-synchronized FAT10-overexpressing cells were released into nocodazole for more than 4 h. Nonetheless, when these double thymidine-treated cells were released into media, a similar number of G1 parental and FAT10-overexpressing HCT116 cells was observed throughout the 10-h time course. Additionally, more nocodazole-treated FAT10-overexpressing cells escape mitotic controls and are multinucleate compared with parental cells. Significantly, we observed a higher degree of variability in chromosome number in cells overexpressing FAT10. Hence, our data suggest that high levels of FAT10 protein in cells lead to increased mitotic nondisjunction and chromosome instability, and this effect is mediated by an abbreviated mitotic phase and the reduction in the kinetochore localization of MAD2 during the prometaphase stage of the cell cycle.