Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos

Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition.     

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation

We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.     

Cyclin A and B functions in the early Drosophila embryo.

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. We investigated the relationship between subcellular localization of cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos. During early embryonic cycles, cyclin A was always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B was predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, cyclin B colocalized with metaphase but not anaphase spindle microtubules. We changed maternal gene doses of cyclins A and B to test their functions in preblastoderm embryos. We observed that increasing doses of cyclin B increased cyclin B-Cdk1 activity, which correlated with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles was affected by cyclin A but not cyclin B levels. Taken together, our observations support the hypothesis that cyclin B regulates cytoskeletal changes while cyclin A regulates the nuclear cycles. Varying the relative levels of cyclins A and B uncoupled the cytoskeletal and nuclear events, so we speculate that a balance of cyclins is necessary for proper coordination during these embryonic cycles.     

Mitotic cyclin distribution during maize cell division: Implications for the sequence diversity and function of cyclins in plants

Summary Cyclin proteins are components of the regulatory system that controls the orderly progression of the events of cell division. Their sub-cellular location, as well as their fluctuating abundance and their affinities for the cyclin-dependent kinases (CDKs) to which they bind, determine their successive roles during the cell cycle. Here we employ species-specific antibodies to monitor changes in quantity and location of four maize cyclins and maize Cdc2-kinase in dividing maize root tip cells. Maize cyclin Ia occurs in the nuclear matrix and is released when the nuclear envelope breaks down. In contrast, cyclin Ib is cytoplasmic until prophase; it associates transiently with the nuclear envelope and preprophase band (PPB) just before these structures break down and then associates with the condensed chromosomes and spindle region before declining at anaphase. Cyclin II and Cdc2 also occur in the PPB. Occurrence of cyclin Ib and Cdc2 at the PPB concurrent with initiation of breakdown is consistent with previous studies in which microinjection of cyclin-dependent protein kinase indicated that removal of the PPB at the time of nuclear-envelope breakdown is catalysed by a CDK. While cyclins Ia and III are predominantly nuclear prior to mitosis, cyclins Ib and II are predominantly cytoplasmic until prophase then become nuclear. The initial cytoplasmic retention of cyclins Ib and II correlates with their possession of a sequence similar to the cytoplasmic-retention signal of animal cyclin B1. Cyclin II binds to all microtubule arrays during the cell cycle, becoming markedly concentrated in the phragmoplast, and cyclin III associates with the spindle and then the phragmoplast. Cdc2 also occurs in the phragmoplast. Persistence of mitotic cyclins and CDK after mitosis into the cytokinetic stage, as seen in maize, is not paralleled in animal cells, where the cytokinetic mid-body is not so labelled, presumably reflecting the key role of the phragmoplast apparatus in plant cell division.Abbreviations CDK cyclin-dependent kinase - CRS cytoplasmicretention signal - NE nuclear envelope - NEB nuclear-envelope breakdown - NLS nuclear-location signal - PPB preprophase band - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate     

Redistribution of nuclear lamins in mitotic cells

The nuclear lamins are directed from the cytoplasm to chromosomes as part of the maturation pathway of the interphase nucleoskeleton. In mitosis, the three polypeptides lamin A, B and C were found in the cytoplasm from prophase until anaphase and shifted to chromosomal surfaces at telophase (Ely, D'Arcy and Jost, 1978; Gerace, Blum and Blobel, 1978). We show here that early events in nucleoskeleton formation could be regulated by extracellular pH. When exponentially growing tissue culture cells and cells arrested in mitosis were exposed to different extracellular pH values, three patterns of distribution of lamins were observed in mitotic cells: exclusively cytoplasmic distribution of mitotic lamins at low pH (6.8 to 7.3); a premature association of a lamin subfraction with metaphase chromosomes at intermediate pH 7.5; a more prominent relocation of lamins onto chromosomes in metaphase and in disorganized metaphase at pH 8.0. Reassembly of lamins occurred at telomeric ends of mitotic chromosomes followed by a lateral fusion to form a nuclear cage. Using immunogold localization, we show that pH-induced, premature, partial deposition of lamins onto condensed chromosomes may occur prior to the formation of the bilamellar nuclear envelope. These results suggest that the pH-induced redistribution of lamins acts to trigger early events of mitosis to interphase transition.     

Expression and function of Drosophila cyclin A during embryonic cell cycle progression

Cyclin proteins are thought to trigger entry into mitosis. During mitosis they are rapidly degraded. Therefore, mitosis and consequently cyclin degradation might be triggered at a time when cyclins have reaccumulated to a critical level. We cloned and sequenced a Drosophila cyclin A homolog and identified mutations in the corresponding gene. Immunofluorescent staining revealed that cyclin A accumulates in the interphase cytoplasm of cellularized embryos, but relocates to the nuclear region early in prophase and is completely degraded within metaphase. Cyclin A was expressed in dividing cells throughout development, and a functional cyclin A gene was required for continued division after exhaustion of maternally contributed cyclin A. Importantly, the timing of post cellularization divisions was not governed by the rate of accumulation or level of cyclin A.     

cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells

A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase. Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level. By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase. Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm. During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes. The nuclear locale reappears during telophase. In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T. Hunter. 1991. J. Cell Biol. 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity.     

Cyclin B1 is rate limiting but not essential for mitotic entry and progression in mammalian somatic cells

Cyclin B1 should have some rate limiting function for cell cycle progression. To test this, we measured the effect of siRNA-mediated depletion of cyclin B1 on mitotic entry and timing. We depleted cyclin B1 in HeLa and hTert-RPE1 cells to levels equivalent or below those achieved in the telophase-to-G1 window. Average cyclin B1/Cdk1 activity was measured in HeLa cells and depleted by ~99%. In both cell lines, this caused ~20% increase in the G2 and ~20% increase the M traverse time. However, co-depletion of cyclin B1 and B2 induced a profound increase in G2 cells, a dramatic reduction in mitotic cells, and an increase in a 4C cycling population. We conclude that any residual levels of cyclin B1 were not sufficient to promote stable mitotic entry and transition in absence of normal levels of cyclin B2. Therefore, we conclude that B cyclin is necessary for mitosis but cyclin B1 is not. Nocodazole treated, cyclin B1-depleted HeLa cells arrested but exited that arrest at higher rates than controls, suggesting that the duration of the spindle checkpoint was affected. In B1 depleted cells, population growth was delayed but evidence of cell death was not consistently observed. A strong phenotype of mitotic chromosomal aberration was observed in HeLa cells depleted for either cyclin but not in RPE cells. In B1 or B2 depleted cells, maloriented chromosomes at metaphase were increased 10 fold and one third of affected metaphase cells entered anaphase without congression. Lagging chromosomes at anaphase were dramatically increased. The aggregate evidence from our study and others suggests that the common effect of cyclin B1 depletion is mild cell cycle perturbation. Lack of uniformity in other phenotypes suggest that these are low penetrance effects that are exacerbated or compensated in some systems by other mechanisms.     

Nek2 localizes to multiple sites in mitotic cells, suggesting its involvement in multiple cellular functions during the cell cycle.

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand the possible cellular processes in which Nek2 participates during the cell cycle, we investigated the expression and subcellular localization of Nek2 in mitotic cells. The Nek2 protein levels were observed to be regulated in a cell cycle stage-specific manner in cultured cells. The cell cycle stage specificity of Nek2 expression was also confirmed in cells undergoing mitosis in vivo. Nek2 proteins were localized in both the nucleus and cytoplasm throughout the cell cycle, but exhibited dynamic changes in distribution, depending on the cell cycle stage. Nek2 was associated with chromosomes from prophase to metaphase and then was dissociated upon entering into anaphase. Nek2 then appeared at the midbody of the cytoplasmic bridge at telophase. Nek2 was also associated with the centrosome throughout the cell cycle as observed previously by others. Additionally, the nuclear localization of Nek2 was increased during S phase. Such dynamic behavior of Nek2 suggests that Nek2 may be a mitotic regulator that is involved in diverse cell cycle events.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.     

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.     

Nuclear and microtubular cycles in heterophasic multinuclearTriticum root-tip cells induced by caffeine

Summary Nuclear and microtubular cycles were studied in large heterophasic multinuclear cells induced in root tips ofTriticum turgidum by caffeine treatment. Multinuclear cells and cells with polyploid nuclei exhibited various configurations of multiple and complex preprophase microtubule (Mt) bands (PPBs), including helical ones. The developmental stages of PPBs in some heterophasic cells did not comply with the cell cycle stages of the associated nuclei, a fact indicating that these events are not directly controlled by the associated nuclei. The heterophasic cells exhibited asynchronous nuclei at different stages of mitosis. In cells displaying prophase and interphase nuclei, the prophase spindle was either absent or developed around both of them or developed around the prophase nuclei earlier than around the interphase ones. During prometaphase-metaphase of the advanced nuclei the lagging interphase nuclei were induced to form prematurely condensed chromosomes (PCCs) along with spindle formation around them. These observations suggest that the mitotic transition in heterophasic cells is delayed but is ultimately achieved due to the effect of the advanced nuclei, which induces a premature mitotic entry of the lagging nuclei. Although kinetochore Mt bundles were found associated with PCCs, their metaphase and anaphase spindles were abnormal resulting in abnormal or abortive anaphases. In some heterophasic cells, metaphase-anaphase transition did not take place simultaneously in different chromosome groups, signifying that the cells do not exit from the mitotic state after anaphase initiation of the advanced nuclei. Asynchronous pace of mitosis of different chromosome groups was also observed during anaphase and telophase. Implications of these observations in understanding plant cell cycle regulation are discussed.Abbreviations cdk cyclin dependent kinase - Mt microtubule - PCC prematurely condensed chromosome - PPB preprophase band     

Inactivation of Cdk1/Cyclin B in metaphase-arrested mouse FT210 cells induces exit from mitosis without chromosome segregation or cytokinesis and allows passage through another cell cycle

It is well known that inactivation of Cdk1/Cyclin B is required for cells to exit mitosis. The work reported here tests the hypothesis that Cdk1/Cyclin B inactivation is not only necessary but also sufficient to induce mitotic exit and reestablishment of the interphase state. This hypothesis predicts that inactivation of Cdk1 in metaphase-arrested cells will induce the M to G1-phase transition. It is shown that when mouse FT210 cells (in which Cdk1 is temperature-sensitive) are arrested in metaphase and then shifted to their non-permissive temperature, they rapidly exit mitosis as evidenced by reassembly of interphase nuclei, decondensation of chromosomes, and dephosphorylation of histones H1 and H3. The resulting interphase cells are functionally normal as judged by their ability to progress through another cell cycle. However, they have double the normal number of chromosomes because they previously bypassed anaphase, chromosome segregation, and cytokinesis. These results, taken together with other observations in the literature, strongly suggest that in mammalian cells, inactivation of Cdk1/cyclin B is the trigger for mitotic exit and reestablishment of the interphase state.     

Requirements of cyclin a for mitosis are independent of its subcellular localization

Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. We show that interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Here, we tested if the unique mitotic function of CycA requires nuclear accumulation. We fused subcellular localization signals to CycA and tested their mitotic capability. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA was able to induce mitosis. We noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16(th) embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein.     

Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus.

We have raised and characterized antibodies specific for human cyclin B2 and have compared the properties of cyclins B1 and B2 in human tissue culture cells. Cyclin B1 and B2 levels are very low in G1 phase, increase in S and G2 phases and peak at mitosis. Both B-type cyclins associate with p34cdc2; their associated kinase activities appear when cells enter mitosis and disappear as the cyclins are destroyed in anaphase. However, human cyclins B1 and B2 differ dramatically in their subcellular localization. Cyclin B1 co-localizes with microtubules, whereas cyclin B2 is primarily associated with the Golgi region. In contrast to cyclin B1, cyclin B2 does not relocate to the nucleus at prophase, but becomes uniformly distributed throughout the cell. The different subcellular locations of human cyclins B1 and B2 implicate them in the reorganization of different aspects of the cellular architecture at mitosis and indicate that different mitotic cyclin-cyclin-dependent kinase complexes may have distinct roles in the cell cycle.     

Localization and structure of snRNPs during mitosis. Immunofluorescent and biochemical studies

The distribution of U snRNAs during mitosis was studied by indirect immunofluorescence microscopy with snRNA cap-specific anti-m3G antibodies. Whereas the snRNAs are strictly nuclear at late prophase, they become distributed in the cell plasm at metaphase and anaphase. They re-enter the newly formed nuclei of the two daughter cells at early telophase, producing speckled nuclear fluorescent patterns typical of interphase cells. While the snRNAs become concentrated at the rim of the condensing chromosomes and at interchromosomal regions at late prophase, essentially no association of the snRNAs was observed with the condensed chromosomes during metaphase and anaphase. Independent immunofluorescent studies with anti-(U1)RNP autoantibodies, which react specifically with proteins unique to the U1 snRNP species, showed the same distribution of snRNP antigens during mitosis as was observed with the snRNA-specific anti-m3G antibody. Immunoprecipitation studies with anti-(U1)RNP and anti-Sm autoantibodies, as well as protein analysis of snRNPs isolated from extracts of mitotic cells, demonstrate that the snRNAs remain associated in a specific manner with the same set of proteins during interphase and mitosis. The concept that the overall structure of the snRNPs is maintained during mitosis also applies to the coexistence of the snRNAs U4 and U6 in a single ribonucleoprotein complex. Particle sedimentation studies in sucrose gradients reveal that most of the snRNPs present in sonicates of mitotic cells do not sediment as free RNP particles, but remain associated with high molecular weight (HMW) structures other than chromatin, most probably with hnRNA/RNP.     

Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells

Cyclin proteins form complexes with members of the p34cdc2 kinase family and they are essential components of the cell cycle regulatory machinery. They are thought to determine the timing of activation, the subcellular distribution, and/or the substrate specificity of cdc2-related kinases, but their precise mode of action remains to be elucidated. Here we report the cloning and sequencing of avian cyclin B2. Based on the use of monospecific antibodies raised against bacterially expressed protein, we also describe the subcellular distribution of cyclin B2 in chick embryo fibroblasts and in DU249 hepatoma cells. By indirect immunofluorescence microscopy we show that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase. Finally, we have examined the phenotypic consequences of expressing wild-type and mutated versions of avian cyclin B2 in HeLa cells. We found that expression of cyclin B2 carrying a mutation at arginine 32 (to serine) caused HeLa cells to arrest in a pseudomitotic state. Many of the arrested cells displayed multiple mitotic spindles, suggesting that the centrosome cycle had continued in spite of the cell cycle arrest.     

Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition

Activation of Cdc2-cyclin B (or M phase-promoting factor (MPF)) at the prophase/metaphase transition proceeds in two steps: dephosphorylation of Cdc2 and phosphorylation of cyclin B. We here investigated the regulation of cyclin B phosphorylation using the starfish oocyte model. Cyclin B phosphorylation is not required for Cdc2 kinase activity; both the prophase complex dephosphorylated on Cdc2 with Cdc25 and the metaphase complex dephosphorylated on cyclin B with protein phosphatase 2A display high kinase activities. An in vitro assay of cyclin B kinase activity closely mimics in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K3, a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.     

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.