The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis

BACKGROUND: Degradation of the mitotic cyclins is a hallmark of the exit from mitosis. Induction of stable versions of each of the three mitotic cyclins of Drosophila, cyclins A, B, and B3, arrests mitosis with different phenotypes. We tested a recent proposal that the destruction of the different cyclins guides progress through mitosis. RESULTS: Real-time imaging revealed that arrest phenotypes differ because each stable cyclin affects specific mitotic events differently. Stable cyclin A prolonged or blocked chromosome disjunction, leading to metaphase arrest. Stable cyclin B allowed the transition to anaphase, but anaphase A chromosome movements were slowed, anaphase B spindle elongation did not occur, and the monooriented disjoined chromosomes began to oscillate between the spindle poles. Stable cyclin B3 prevented normal spindle maturation and blocked major mitotic exit events such as chromosome decondensation but nonetheless allowed chromosome disjunction, anaphase B, and formation of a cytokinetic furrow, which split the spindle. CONCLUSIONS: We conclude that degradation of distinct mitotic cyclins is required to transit specific steps of mitosis: cyclin A degradation facilitates chromosome disjunction, cyclin B destruction is required for anaphase B and cytokinesis and for directional stability of univalent chromosome movements, and cyclin B3 degradation is required for proper spindle reorganization and restoration of the interphase nucleus. We suggest that the schedule of degradation of cyclin A, cyclin B, and then cyclin B3 contributes to the temporal coordination of mitotic events.     

The yeast mitotic cyclin Clb2 cannot substitute for S phase cyclins in replication origin firing

Cyclin-dependent kinases (CDKs) drive the cell cycle, central to which is the accurate control of chromosome replication. In Saccharomyces cerevisiae, six closely related B-type cyclins (Clb1–6) drive the events of S phase and mitosis. Either Clb5 or Clb6 can activate early-firing replication origins, whereas only Clb5 can activate late origins. Clb1–4 are expressed later in the cell cycle. Whether Clb cyclins differ only in timing of expression, or else impart different kinase specificities is under ongoing investigation. This study shows that the expression of Clb2 during S phase in cells lacking Clb5 failed to rescue late origin activation. Early expression of Clb2 in cells lacking both Clb5 and Clb6 did not activate early origins on schedule to restore the correct S phase entry time. Therefore, Clb2 cannot drive timely activation of either early or late replication origins, demonstrating that Clb2-directed CDK has a specificity distinct from that driven by Clb5 and Clb6.     

The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae.

cdc28-1N is a conditional allele that has normal G1 (Start) function but confers a mitotic defect. We have isolated seven genes that in high dosage suppress the growth defect of cdc28-1N cells but not of Start-defective cdc28-4 cells. Three of these (CLB1, CLB2, and CLB4) encode proteins strongly homologous to G2-specific B-type cyclins. Another gene, CLB3, was cloned using PCR, CLB1 and CLB2 encode a pair of closely related proteins; CLB3 and CLB4 encode a second pair. Neither CLB1 nor CLB2 is essential; however, disruption of both is lethal and causes a mitotic defect. Furthermore, the double mutant cdc28-1N clb2::LEU2 is nonviable, whereas cdc28-4 clb2::LEU2 is viable, suggesting that the cdc28-1N protein may be defective in its interaction with B-type cyclins. Our results are consistent with CDC28 function being required in both G1 and mitosis. Its mitotic role, we believe, involves interaction with a family of at least four G2-specific cyclins.     

Bistability of mitotic entry and exit switches during open mitosis in mammalian cells

Mitotic entry and exit are switch‐like transitions that are driven by the activation and inactivation of Cdk1 and mitotic cyclins. This simple on/off reaction turns out to be a complex interplay of various reversible reactions, feedback loops, and thresholds that involve both the direct regulators of Cdk1 and its counteracting phosphatases. In this review, we summarize the interplay of the major components of the system and discuss how they work together to generate robustness, bistability, and irreversibility. We propose that it may be beneficial to regard the entry and exit reactions as two separate reversible switches that are distinguished by differences in the state of phosphatase activity, mitotic proteolysis, and a dramatic rearrangement of cellular components after nuclear envelope breakdown, and discuss how the major Cdk1 activity thresholds could be determined for these transitions.     

The cytoplasmic origin of variability in the timing of S phase in mammalian cells.

The time at which S phase begins in mammalian cells is highly variable with respect to cell age. Evidence is presented that this variability does not arise because the initiation of DNA synthesis depends on the stochastic interaction of an initiator substance with a rare initiation site. Instead, the signal responsible for starting S phase must appear at random in the cytoplasm and may be transient.     

The vesicular compartment of the mitotic apparatus in mammalian cells

Intracellular membranes might play an eminent role in regulating several events during mitosis: In this paper the appearance and changing configurations of the vesicular compartment of the mitotic apparatus of HeLa cells was studied from anaphase to telophase. In early prophase electron opaque and transparent membranous vesicles are found in the pericentriolar region outside the nucleus. During prometaphase when the nuclear envelope opens and starts to disappear, an increasing number of these vesicles appears in the mitotic apparatus near the chromosomes. During metaphase vesicles are spread all over the mitotic apparatus, the number of electron opaque vesicles decreases while the total amount of vesicles does not change significantly. Anaphase shows the same pattern of distribution in the half-spindle and in the midbody. In telophase the amount of electron opaque vesicles increases again. They are now found around vacuoles and near the newly appearing Golgi-cisternae. We assume that the electron opaque vesicles are derived from the Golgi- apparatus which disintegrates during prophase and reappears in late telophase. The change in the appearance of the different types of vesicles during metaphase coincides with drastic changes in the ionic milieu in the mitotic apparatus (Wolniak et al., 1983).     

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.     

The end of mitosis from a phosphatase perspective

Transition through mitosis, the cell division cycle phase deputed to segregate replicated chromosomes, requires a wave of protein phosphorylation. While in the past decades a wealth of information has been gathered on the major kinase activities responsible for the onset of mitosis, only recently has a picture emerged of how their effects are reversed by protein phosphatases at the end of mitosis. Here, we summarized some recent data on the relevance for protein phosphatases in the reversal of mitotic phosphorylation required to complete mitosis in vertebrate cells.     

The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.     

Cyclin-dependent kinases and S phase control in mammalian cells

Mammalian DNA replication is an elegantly choreographed process in which multiple components are assembled at the origins to form the prereplication complex. Formation and activation of the prereplication complex requires coordinate actions of G1and S phase cyclin-dependent kinases. Cyclin E-CDK2 and cyclin A-CDK2, together with DBF4-CDC7, phosphorylate several components of the prereplication complex and replication machinery. In this review, we summarize the current understanding of the mechanism of initiation of DNA replication in mammalian cells. The roles of cyclin A/E-CDK2 complexes in driving replication, their relationship with other regulators of S phase, and their role in keeping replication to only once per cell cycle will be discussed. In addition, an important issue is the checks and balances that prevent inappropriate DNA replication, and how a breakdown in these checkpoints can lead to genomic instability and cancer. A critical mediator of these checkpoints, ATM, signals through a comprehensive network of proteins leading to CDK2 inhibition thus preventing DNA synthesis. This will be reviewed in addition to other mechanisms involved in the intra-S phase DNA damage checkpoint.     

Staurosporine overrides checkpoints for mitotic onset in BHK cells.

Under normal conditions, mammalian cells will not initiate mitosis in the presence of either unreplicated or damaged DNA. We report here that staurosporine, a tumor promoter and potent protein kinase inhibitor, can uncouple mitosis from the completion of DNA replication and override DNA damage-induced G2 delay. Syrian hamster (BHK) fibroblasts that were arrested in S phase underwent premature mitosis at concentrations as low as 1 ng/ml, with maximum activity seen at 50 ng/ml. Histone H1 kinase activity was increased to approximately one-half the level found in normal mitotic cells. Inhibition of protein synthesis during staurosporine treatment blocked premature mitosis and suppressed the increase in histone H1 kinase activity. In asynchronously growing cells, staurosporine transiently increased the mitotic index and histone H1 kinase activity but did not induce S phase cells to undergo premature mitosis, indicating a requirement for S phase arrest. Staurosporine also bypassed the cell cycle checkpoint that prevents the onset of mitosis in the presence of damaged DNA. The delay in mitotic onset resulting from gamma radiation was reduced when irradiation was followed immediately by exposure to 50 ng/ml of staurosporine. These findings indicate that inhibition of protein phosphorylation by staurosporine can override two important checkpoints for the initiation of mitosis in BHK cells.     

Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).     

Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis

The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF(CDC4) from the end of G1phase until mitosis in the budding yeast Saccharomyces cerevisiae. Here we describe a dominant-negative CDC6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more efficiently than wild-type Cdc6, in part because it is completely refractory to SCF(CDC4)-mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. We show that this site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identified previously near the N-terminus of the protein. We show that both sites can target Cdc6 for proteolysis in late G1/early S phase whilst only the newly identified site can target Cdc6 for proteolysis during mitosis.     

Golgi membranes remain segregated from the endoplasmic reticulum during mitosis in mammalian cells

What happens to organelles during mitosis, and how they are apportioned to each of the daughter cells, is not completely clear. We have devised a procedure to address whether Golgi membranes fuse with the Endoplasmic Reticulum (ER) during mitosis via the detection of interactions between ER and Golgi proteins. This procedure involves coexpressing an FKBP-tagged Golgi enzyme with an ER-retained protein fused to FRAP in COS cells. Since treatment with rapamycin induces a tight association between FKBP and FRAP, one would expect rapamycin to trap the FKBP-fused Golgi protein in the ER if it ever visits the ER during mitosis. However, after the doubly transfected cells progress through mitosis in the presence of rapamycin, we find the Golgi protein in the newly formed Golgi stacks and not in the ER. Based on these results, we conclude that Golgi membranes remain separate from the ER during mitosis in mammalian cells.     

The Drosophila RAD21 cohesin persists at the centromere region in mitosis

'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in [1,2]). Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins [3]. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition. We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition.     

Changes in the rate of DNA replication fork movement during S phase in mammalian cells.

DNA fiber autoradiography was used to measure the rate of replication fork movement and the size of replication units as a function of time during the S phase of synchronized Chinese hamster ovary cells. The rate of fork movement increased by about threefold from early S to later S phase, with the most dramatic change occurring in the first hour of S phase. On the other hand, the size of replication units did not vary significantly during S phase.