Calcium and cell cycle control

The cell division cycle of the early sea urchin embryo is basic. Nonetheless, it has control points in common with the yeast and mammalian cell cycles, at START, mitosis ENTRY and mitosis EXIT. Progression through each control point in sea urchins is triggered by transient increases in intracellular free calcium. The Cai transients control cell cycle progression by translational and post-translational regulation of the cell cycle control proteins pp34 and cyclin. The START Cai transient leads to phosphorylation of pp34 and cyclin synthesis. The mitosis ENTRY Cai transient triggers cyclin phosphorylation. The motosis EXIT transient causes destruction of phosphorylated cyclin. We compare cell cycle regulation by calcium in sea urchin embryos to cell cycle regulation in other eggs and oocytes and in mammalian cells.     

Processive ubiquitin chain formation by the anaphase-promoting complex

Progression through mitosis requires the sequential ubiquitination of cell cycle regulators by the anaphase-promoting complex, resulting in their proteasomal degradation. Although several mechanisms contribute to APC/C regulation during mitosis, the APC/C is able to discriminate between its many substrates by exploiting differences in the processivity of ubiquitin chain assembly. Here, we discuss how the APC/C achieves processive ubiquitin chain formation to trigger the sequential degradation of cell cycle regulators during mitosis.     

Cell‐Cycle‐Dependent Regulation of Translation: New Interpretations of Old Observations in Light of New Approaches

It is a long‐standing view that global translation varies during the cell cycle and is much lower in mitosis than in other cell‐cycle phases. However, the central papers in the literature are not in agreement about the extent of downregulation in mitosis, ranging from a dramatic decrease to only a marginal reduction. Herein, it is argued that the discrepancy derives from technical challenges. Cell‐cycle‐dependent variations are most conveniently studied in synchronized cells, but the synchronization methods by themselves often evoke stress responses that, in turn, affect translation rates. Further, it is argued that previously reported cell‐cycle‐dependent changes in the global translation rate to a large extent reflect responses to the synchronization methods. Recent findings strongly suggest that the global translation rate is not regulated in a cell‐cycle‐dependent manner. Novel techniques allowing a genome‐wide analysis of translational profiles suggest that the extent and importance of selective translational regulation associated with cell‐cycle transitions have been underestimated. Therefore, the main question is which messenger RNAs (mRNAs) are translated, rather than whether the global translation rate is decreased.     

Actin cytoskeleton dynamics and the cell division cycle

The network of actin filaments is one of the crucial cytoskeletal structures contributing to the morphological framework of a cell and which participates in the dynamic regulation of cellular functions. In adherent cell types, cells adhere to the substratum during interphase and spread to assume their characteristic shape supported by the actin cytoskeleton. This actin cytoskeleton is reorganized during mitosis to form rounded cells with increased cortical rigidity. The actin cytoskeleton is re-established after mitosis, allowing cells to regain their extended shape and attachment to the substratum. The modulation of such drastic changes in cell shape in coordination with cell cycle progression suggests a tight regulatory interaction between cytoskeleton signalling, cell–cell/cell–matrix adhesions and mitotic events. Here, we review the contribution of the actin cytoskeleton to cell cycle progression with an emphasis on the effectors responsible for the regulation of the actin cytoskeleton and integration of their activities with the cell cycle machinery.     

Translational control of the embryonic cell cycle

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells.     

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.     

Gene expression regulation at the translational level: contribution of marine organisms

Gene expression regulation is crucial for organism survival. Each step has to be regulated, from the gene to the protein. mRNA can be stored in the cell without any direct translation. This process is used by the cell to control protein synthesis rapidly at the right place, at the right time. Protein synthesis costs a lot of energy for the cell, so that a precise control of this process is required. Translation initiation represents an important step to regulate gene expression. Many factors that can bind mRNA and recruit different partners are involved in the inhibition or stimulation of protein synthesis. Oceans contain an important diversity of organisms that are used as important models to analyse gene expression at the translational level. These are useful to study translational control in different physiological processes for instance cell cycle (meiosis during meiotic maturation of starfish oocytes, mitosis following fertilization of sea urchin eggs) or to understand nervous system mechanisms (aplysia). All these studies will help finding novel actors involved in translational control and will thus be useful to discover new targets for therapeutic treatments against human diseases.     

Identification of c-Fos as a mitotic phosphoprotein: regulation of c-Fos by Aurora-A

The c-Fos has been implicated in the regulation of gene expression under a variety of stimuli. It is known that c-Fos undergoes protein phosphorylation, which may subsequently modulate diverse functions in cells. However, less is known about the role and phosphorylation status of c-Fos during mitosis. Here, we showed that c-Fos exhibited an electrophoretic mobility up-shift as detected by SDS-PAGE during mitosis, which is an indication of protein phosphorylation. Aurora-A, but not Aurora-B or -C, serves as one of the kinases catalyzing the mitotic phosphorylation of c-Fos. The mobility up-shift was partially abolished by introducing siRNA or a catalytically inactive form of Aurora-A. Moreover, ectopic expression of the wild type, but not the catalytically inactive form of Aurora-A resulted in the alteration of c-Fos complex formation, suggesting Aurora-A is engaged in the regulation of c-Fos protein–protein interaction. These findings imply that c-Fos may undergo cell cycle dependent phosphorylation, in which some kinases including Aurora-A play a role in catalyzing the post translational modification of c-Fos. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Who guards the guardian? Mechanisms that restrain APC/C during the cell cycle

The cell cycle is principally controlled by Cyclin Dependent Kinases (CDKs), whose oscillating activities are determined by binding to Cyclin coactivators. Cyclins exhibit dynamic changes in abundance as cells pass through the cell cycle. The sequential, timed accumulation and degradation of Cyclins, as well as many other proteins, imposes order on the cell cycle and contributes to genome maintenance. The destruction of many cell cycle regulated proteins, including Cyclins A and B, is controlled by a large, multi-subunit E3 ubiquitin ligase termed the Anaphase Promoting Complex/Cyclosome (APC/C). APC/C activity is tightly regulated during the cell cycle. Its activation state increases dramatically in mid-mitosis and it remains active until the end of G1 phase. Following its mandatory inactivation at the G1/S boundary, APC/C activity remains low until the subsequent mitosis. Due to its role in guarding against the inappropriate or untimely accumulation of Cyclins, the APC/C is a core component of the cell cycle oscillator. In addition to the regulation of Cyclins, APC/C controls the degradation of many other substrates. Therefore, it is vital that the activity of APC/C itself be tightly guarded. The APC/C is most well studied for its role and regulation during mitosis. However, the APC/C also plays a similarly important and conserved role in the maintenance of G1 phase. Here we review the diverse mechanisms counteracting APC/C activity throughout the cell cycle and the importance of their coordinated actions on cell growth, proliferation, and disease.     

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.     

The relationship between levels and rates of synthesis of polyamines during mammalian cell cycle

The objective of this study was to examine the rate of synthesis and the intracellular levels of polyamines as a function of the HeLa cell cycle. The intracellular levels of ornithine, which were high during mitosis and early G1 phase, decreased rapidly during late G1 phase when the ornithine decarboxylase activity was at its peak. The activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase reached a peak during G1 and decreased rapidly during the S phase. The levels of polyamines were maximum in mitosis and S phase. In constrast, the rate of polyamine synthesis during S phase was 5–10 fold lower than that in mitosis or G1 phase. We have also observed fluctuations in diamine-oxidase activity during the cell cycle. The enzyme activity was high during mitosis and late G1 and low during S phase. Thus, the results of this study suggest an important role for the catabolic enzymes in the regulation of polyamine levels during the mammalian cell cycle.     

Fresh WNT into the regulation of mitosis

Canonical Wnt signaling triggering β-catenin-dependent gene expression contributes to cell cycle progression, in particular at the G1/S transition. Recently, however, it became clear that the cell cycle can also feed back on Wnt signaling at the G2/M transition. This is illustrated by the fact that mitosis-specific cyclin-dependent kinases can phosphorylate the Wnt co-receptor LRP6 to prime the pathway for incoming Wnt signals when cells enter mitosis. In addition, there is accumulating evidence that various Wnt pathway components might exert additional, Wnt-independent functions that are important for proper regulation of mitosis. The importance of Wnt pathways during mitosis was most recently enforced by the discovery of Wnt signaling contributing to the stabilization of proteins other than β-catenin, specifically at G2/M and during mitosis. This Wnt-mediated stabilization of proteins, now referred to as Wnt/STOP, might on one hand contribute to maintaining a critical cell size required for cell division and, on the other hand, for the faithful execution of mitosis itself. In fact, most recently we have shown that Wnt/STOP is required for ensuring proper microtubule dynamics within mitotic spindles, which is pivotal for accurate chromosome segregation and for the maintenance of euploidy.     

Heterogenic Final Cell Cycle by Chicken Retinal Lim1 Horizontal Progenitor Cells Leads to Heteroploid Cells with a Remaining Replicated Genome

Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

Tyrosine phosphorylations specific to mitosis in human and hamster cells

Changes in protein tyrosine phosphorylation are known to be important for regulating cell cycle progression. With the aim of identifying new proteins involved in the regulation of mitosis, we used an antibody against phosphotyrosine to analyze proteins from synchronized human and hamster cells. At least seven proteins were found that displayed mitosis-specific tyrosine phosphorylation in HeLa cells (pp165, 205, 240, 250, 270, 290, and ～ 400) and one such protein in hamster BHK cells (pp155). In synchronized HeLa and BHK cells, all proteins except HeLa pp165, pp205, and pp250 were readily detectable only in mitosis. Tyrosine phosphorylation of pp165, pp205, and pp250 was apparent during arrest in S phase, suggesting that cell cycle perturbations can affect the phosphorylation state of some of these proteins. In a related finding in BHK cells, pp155 underwent tyrosine phosphorylation when cells were forced into premature mitosis by caffeine treatment. Only one protein (pp135 in HeLa cells) was found to be dephosphorylated on tyrosine during mitosis. The above findings may prove helpful for isolating new cell cycle proteins that are important for both the normal regulation of mitosis and the mitotic aberrations associated with cell cycle perturbations and chemical treatments.     

Three-step model for condensin activation during mitotic chromosome condensation

Chromosomes undergo a major structural reorganization during mitosis. The first step in this reorganization is the compaction of interphase chromatin into highly condensed mitotic chromosomes. An evolutionarily conserved multi-subunit ATPase, the condensin complex, plays a critical role in establishing chromosome architecture and promoting chromosome condensation in mitosis. How does condensin promote chromosome condensation and how, in turn, is the cell cycle machinery activating or restraining condensin activity during the cell cycle are fundamental questions for cell biology. In this review, we examine the role of post-translational modifications, and in particular multi-site phosphorylation, in the regulation of condensin activity during the cell cycle. Remarkably, inspection of phosphorylation sites identified through multiple proteome-wide mass spectrometry analyses reveals that the phosphorylation landscape of condensin is highly conserved evolutionarily and that several kinases regulate condensin in vivo. This analysis leads us to propose the ultrasensitive-kinase switch model, whereby the phosphorylation of condensin by multiple kinases allows the process of chromosome condensation to be maintained and even increased under fluctuating levels of cyclin-CDK activity during mitosis. Our model reconciles how chromosome condensation might be highly sensitive to low levels of CDK activity in early mitosis and subsequently insensitive to the declining levels CDK activity in late mitosis.     

ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

Cell cycle specific expression and nucleolar localization of human J-domain containing co-chaperon Mrj

J-domain containing co-chaperone Mrj (mammalian relative to DnaJ) has been implicated in diverse cellular functions including placental development and inhibition of Huntingtin mediated cytotoxicity. It has also been shown to interact with keratin intermediate filaments. Since keratins undergo extensive reorganization during cell division, its interactor Mrj might also play an important role in the regulation of cell cycle. In support of this hypothesis, we report the up-regulation of Mrj protein in M-phase of HeLa cells implicating its role in mitosis related activities. The protein is dispersed throughout the cell during late mitosis and is localized in nucleolus during interphase, confirming that the activity of Mrj is regulated by its cell cycle specific expression together with its differential subcellular localization.