The mitotic centromeric protein MEI-S332 and its role in sister-chromatid cohesion

Faithful segregation of sister chromatids during cell division requires properly regulated cohesion between the sister centromeres. The sister chromatids are attached along their lengths, but particularly tightly in the centromeric regions. Therefore specific cohesion proteins may be needed at the centromere. Here we show that Drosophila MEI-S332 protein localizes to mitotic metaphase centromeres. Both overexpression and mutation of MEI-S332 increase the number of apoptotic cells. In mei-S332 mutants the ratio of metaphase to anaphase figures is lower than wild type, but it is higher if MEI-S332 is overexpressed. In chromosomal squashes centromeric attachments appear weaker in mei-S332 mutants than wild type and tighter when MEI-S332 is overexpressed. These results are consistent with MEI-S332 contributing to centromeric sister-chromatid cohesion in a dose-dependent manner. MEI-S332 is the first member identified of a predicted class of centromeric proteins that maintain centromeric cohesion. Received: 11 December 1998; in revised form: 4 August 1999 / Accepted: 13 August 1999     

RBMX: a regulator for maintenance and centromeric protection of sister chromatid cohesion

Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.     

The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2‐7 subcomplex of the replicative Cdc45‐MCM‐GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL. Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.     

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

The hsSsu72 phosphatase is a cohesin‐binding protein that regulates the resolution of sister chromatid arm cohesion

Cohesin is a multiprotein complex that establishes sister chromatid cohesion from S phase until mitosis or meiosis. In vertebrates, sister chromatid cohesion is dissolved in a stepwise manner: most cohesins are removed from the chromosome arms via a process that requires polo‐like kinase 1 (Plk1), aurora B and Wapl, whereas a minor amount of cohesin, found preferentially at the centromere, is cleaved by separase following its activation by the anaphase‐promoting complex/cyclosome. Here, we report that our budding yeast two‐hybrid assay identified hsSsu72 phosphatase as a Rad21‐binding protein. Additional experiments revealed that Ssu72 directly interacts with Rad21 and SA2 in vitro and in vivo, and associates with sister chromatids in human cells. Interestingly, depletion or mutational inactivation of Ssu72 phosphatase activity caused the premature resolution of sister chromatid arm cohesion, whereas the overexpression of Ssu72 yielded high resistance to this resolution. Interestingly, it appears that Ssu72 regulates the cohesion of chromosome arms but not centromeres, and acts by counteracting the phosphorylation of SA2. Thus, our study provides important new evidence, suggesting that Ssu72 is a novel cohesin‐binding protein capable of regulating cohesion between sister chromatid arms.     

Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle

Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This enzyme is a collection of varied heterotrimeric complexes, each composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit. To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of the PP2A subunits of Saccharomyces cerevisiae. We found the following: the level of each subunit remained constant throughout the cell cycle; there is at least 10 times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding. Using green fluorescent protein-tagged forms of each subunit, we monitored the sites of significant accumulation of each protein throughout the cell cycle. The two regulatory subunits displayed distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck, and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Although Rts1p and Tpd3p required heterotrimer formation to achieve normal localization, Cdc55p achieved its normal localization in the absence of either an A or C subunit.     

Regulation of the mitotic exit protein kinases Cdc15 and Dbf2

In budding yeast, the release of the protein phosphatase Cdc14 from its inhibitor Cfi1/Net1 in the nucleolus during anaphase triggers the inactivation of Clb CDKs that leads to exit from mitosis. The mitotic exit pathway controls the association between Cdc14 and Cfi1/Net1. It is comprised of the RAS-like GTP binding protein Tem1, the exchange factor Lte1, the GTPase activating protein complex Bub2-Bfa1/Byr4, and several protein kinases including Cdc15 and Dbf2. Here we investigate the regulation of the protein kinases Dbf2 and Cdc15. We find that Cdc15 is recruited to both spindle pole bodies (SPBs) during anaphase. This recruitment depends on TEM1 but not DBF2 or CDC14 and is inhibited by BUB2. Dbf2 also localizes to SPBs during anaphase, which coincides with activation of Dbf2 kinase activity. Both events depend on the mitotic exit pathway components TEM1 and CDC15. In cells lacking BUB2, Dbf2 localized to SPBs in cell cycle stages other than anaphase and telophase and Dbf2 kinase was prematurely active during metaphase. Our results suggest an order of function of mitotic exit pathway components with respect to SPB localization of Cdc15 and Dbf2 and activation of Dbf2 kinase. BUB2 negatively regulates all 3 events. Loading of Cdc15 on SPBs depends on TEM1, whereas loading of Dbf2 on SPBs and activation of Dbf2 kinase depend on TEM1 and CDC15.     

A REC8-dependent plant Shugoshin is required for maintenance of centromeric cohesion during meiosis and has no mitotic functions

During meiosis, sequential release of sister chromatid cohesion (SSC) during two successive nuclear divisions allows the production of haploid gametes from diploid progenitor cells. Release of SSC along chromosome arms allows first a reductional segregation of homologs, and, subsequently, release of centromeric cohesion at anaphase II allows the segregation of chromatids. The Shugoshin (SGO) protein family plays a major role in the protection of centromeric cohesion in Drosophila and yeast. We have isolated a maize mutant that displays premature loss of centromeric cohesion at anaphase I. We showed that this phenotype is due to the absence of ZmSGO1 protein, a maize shugoshin homolog. We also show that ZmSGO1 is localized to the centromeres. The ZmSGO1 protein is not found on mitotic chromosomes and has no obvious mitotic function. On the basis of these results, we propose that ZmSGO1 specifically maintains centromeric cohesion during meiosis I and therefore suggest that SGO1 core functions during meiosis are conserved across kingdoms and in large-genome species. However, in contrast to other Shugoshins, we observed an early and REC8-dependent recruitment of ZmSGO1 in maize, suggesting that control of SGO1 recruitment to chromosomes is different in plants than in other model organisms.     

The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.     

A matter of choice: the establishment of sister chromatid cohesion

Sister chromatid cohesion is the basis for the recognition of chromosomal DNA replication products for their bipolar segregation in mitosis. Fundamental to sister chromatid cohesion is the ring‐shaped cohesin complex, which is loaded onto chromosomes long before the initiation of DNA replication and is thought to hold replicated sister chromatids together by topological embrace. What happens to cohesin when the replication fork approaches, and how cohesin recognizes newly synthesized sister chromatids, is poorly understood. The characterization of a number of cohesion establishment factors has begun to provide hints as to the reactions involved. Cohesin is a member of the evolutionarily conserved family of Smc subunit‐based protein complexes that contribute to many aspects of chromosome biology by mediating long‐range DNA interactions. I propose that the establishment of cohesion equates to the selective stabilization of those cohesin‐mediated DNA interactions that link sister chromatids in the wake of replication forks.     

The Yin and Yang of redox regulation

Abstract

Mammalian cells produce reactive oxygen and nitrogen species (ROS/RNOS) in response to an oxidative environment. Powerful antioxidant mechanisms have been developed in order to avoid oxidative stress by contributing to the maintenance of redox homeostasis. Traditionally, accumulation of ROS/RNOS is considered deleterious for cells as it can lead to loss of cellular function, aging, and cell death. Consequently, ROS/RNOS imbalance has been implicated in the etiology and/or progression of numerous pathologies such as cardiovascular diseases, inflammation, and cancer. An interesting concept that has emerged more recently is that not only have cells developed efficient systems to cope with ROS/RNOS accumulation but they have also learned to profit of them under certain circumstances. This notion is supported by data showing that ROS/RNOS can act as signaling molecules affecting the function and activity of a multiplicity of protein kinases and phosphatases controlling cellular homeostasis. This review does not provide an exhaustive overview of molecular mechanisms linked to ROS/RNOS generation and processing but includes relevant examples highlighting the dichotomic nature of these small molecules and the multitude of effects elicited by their accumulation. This aspect of ROS/RNOS ought to be taken into account particularly in novel therapeutic setups that aim to achieve high efficiency and minimal or no side effects.     

Characterization of protein kinases in mitotic and meiotic cell extracts

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.     

Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt

A key regulator of many kinase cascades, heterotrimeric protein serine/threonine phosphatase 2A (PP2A), is composed of catalytic (C), scaffold (A), and variable regulatory subunits (B, B', B' gene families). In neuronal PC12 cells, PP2A acts predominantly as a gatekeeper of extracellular signal-regulated kinase (ERK) activity, as shown by inducible RNA interference of the Aalpha scaffolding subunit and PP2A inhibition by okadaic acid. Although okadaic acid potentiates Akt/protein kinase B and ERK phosphorylation in response to epidermal, basic fibroblast, or nerve growth factor, silencing of Aalpha paradoxically has the opposite effect. Epidermal growth factor receptor Tyr phosphorylation was unchanged following Aalpha knockdown, suggesting that chronic Akt and ERK hyperphosphorylation leads to compensatory down-regulation of signaling molecules upstream of Ras and blunted growth factor responses. Inducible exchange of wild-type Aalpha with a mutant with selective B' subunit binding deficiency implicated PP2A/B' heterotrimers as Akt modulators. Conversely, silencing of the B-family regulatory subunits Balpha and Bdelta led to hyperactivation of ERK stimulated by constitutively active MEK1. In vitro dephosphorylation assays further support a role for Balpha and Bdelta in targeting the PP2A heterotrimer to dephosphorylate and inactivate ERKs. Thus, receptor tyrosine kinase signaling cascades leading to Akt and ERK activation are modulated by PP2A holoenzymes with distinct regulatory properties.