MSA-35: a protein identified by human autoantibodies that colocalizes with microtubules.

We have identified a putative 35-kilodalton protein that colocalizes with microtubules and displays a unique spatial and temporal distribution during the cell cycle of HeLa cells. This protein has been given the designation MSA-35. MSA-35 first appears in association with microtubules and centrosomes of interphase cells exhibiting centrosome separation as a prelude to cell division. This protein is found in conjunction with kinetochore microtubules throughout their appearance. MSA-35 transiently associates with interpolar microtubules following anaphase and the pattern of MSA-35 reactivity in telophase cells suggests that there are at least seven domains within the intercellular bridge. The distribution of MSA-35 during and following recovery from mitotic arrest with nocodazole suggest that it is also present at low levels in interphase cells, can associate with interphase centrosomes, and colocalizes with nascent microtubules. The complex spatial and temporal distribution of MSA-35 indicates that it may be necessary for a series of events in the mitotic process such as the bundling of microtubules.     

Nuclear asynchrony in multinucleate rat kangaroo cells

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.     

Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.     

Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren

In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally, brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.     

A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation

Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.     

Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes

The dramatic condensation of chromosomes that occurs during mitosis is widely thought to be largely controlled by a protein complex termed condensin. Here, we describe a conditional knockout of the condensin subunit ScII/SMC2 in chicken DT40 cells. In cells lacking this condensin subunit, chromosome condensation is delayed, but ultimately reaches near-normal levels. However, these chromosomes are structurally compromised. Kinetochores appear normal, but the localization of nonhistone proteins such as topoisomerase II and INCENP is aberrant. Both proteins also fail to partition into the chromosome scaffold fraction, which appears to be largely missing in the absence of condensin. Furthermore, the chromosomes lack structural integrity, as defined by an assay that tests the stability of the chromosomal higher-order structure. Thus, a major function of condensin is to promote the correct association of nonhistone proteins with mitotic chromosomes, and this is essential for establishment of a robust chromosome structure.     

A kinase-anchoring protein (AKAP)95 recruits human chromosome-associated protein (hCAP)-D2/Eg7 for chromosome condensation in mitotic extract

Association of the condensin multiprotein complex with chromatin is required for chromosome condensation at mitosis. What regulates condensin targeting to chromatin is largely unknown. We previously showed that the nuclear A kinase-anchoring protein, AKAP95, is implicated in chromosome condensation. We demonstrate here that AKAP95 acts as a targeting protein for human chromosome-associated protein (hCAP)-D2/Eg7, a component of the human condensin complex, to chromosomes. In HeLa cell mitotic extract, AKAP95 redistributes from the nuclear matrix to chromatin. When association of AKAP95 with chromatin is prevented, the chromatin does not condense. Condensation is rescued by a recombinant AKAP95 peptide containing the 306 COOH-terminal amino acids of AKAP95. Recombinant AKAP95 binds chromatin and elicits recruitment of Eg7 to chromosomes in a concentration-dependent manner. Amount of Eg7 recruited correlates with extent of chromosome condensation: resolution into distinct chromosomes is obtained only when near-endogenous levels of Eg7 are recruited. Eg7 and AKAP95 immunofluorescently colocalize to the central region of methanol-fixed metaphase chromosomes. GST pull-down data also suggest that AKAP95 recruits several condensin subunits. The results implicate AKAP95 as a receptor that assists condensin targeting to chromosomes.     

Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes

DNA methylation patterns were studied at the chromosome level in normal and abnormal X chromosomes using an anti-5-methylcytosine antibody. In man, except for the late-replicating X of female cells, the labeled chromosome structures correspond to R- and T-bands and heterochromatin. Depending on the cell type, the species, and cell culture conditions, the late-replicating X in female cells appears to be more or less undermethylated. Under normal conditions, the only structures that remain methylated on the X chromosomes correspond to pseudoautosomal regions, which harbor active genes. Thus, active genes are usually hypomethylated but are located in methylated chromatin. Structural rearrangements of the X chromosome, such as t(X;X)(pter;pter), induce a Turner syndrome-like phenotype that is inconsistent with the resulting triple-X constitution. This suggests a position effect controlling gene inactivation. The derivative chromosomes are always late replicating, and their duplicated short arms, which harbor pseudoautosomal regions, replicate later than the normal late-replicating X chromosomes. The compaction or condensation of this segment is unusual, with a halo of chromatin surrounding a hypocondensed chromosome core. The chromosome core is hypomethylated, but the surrounding chromatin is slightly labeled. Thus, unusual DNA methylation and chromatin condensation are associated with the observed position effect. This strengthens the hypothesis that DNA methylation at the chromosome level is associated with both chromatin structure and gene expression.     

Monoclonal antibody with specificity to mitotic chromosomes of primates

Fusion of a cell in mitosis with a cell in interphase results in the condensation of chromatin in the interphase nucleus into chromosomes. Premature chromosome condensation is caused by certain proteins, called mitotic factors, that are present in the mitotic cell and are localized on chromosomes. Extracts from mitotic cells were used to immunize mice to produce monoclonal antibodies specific for cells in mitosis. Among the antibodies obtained, the MPM-4 antibody defines a 125-kD polypeptide antigen located on mitotic chromosomes by indirect immunofluorescence. Although the polypeptide antigen is present in approximately equal concentrations in extracts of interphase cells and mitotic cells, as revealed by immunoblots, it cannot be detected cytologically in the former. Cell fractionation experiments showed that the 125-kD antigen is found in the cytoplasm of interphase cells and metaphase cells, but is concentrated in fractions containing metaphase chromosomes, although not detectable in interphase nuclei. Even though the antigen is apparently primate-specific, it binds to mitotic chromosomes and prematurely condensed chromosomes in human-rodent cell hybrids without regard to the species of origin of the mitotic inducer. The presence of the antigen in the cytoplasm of interphase cells and the chromosomes of mitotic cells suggests a relationship between the presence of the antigen on chromosomes and the process of chromosome condensation and decondensation.     

Bacillus subtilis SMC is required for proper arrangement of the chromosome and for efficient segregation of replication termini but not for bipolar movement of newly duplicated origin regions

SMC protein is required for chromosome condensation and for the faithful segregation of daughter chromosomes in Bacillus subtilis. The visualization of specific sites on the chromosome showed that newly duplicated origin regions in growing cells of an smc mutant were able to segregate from each other but that the location of origin regions was frequently aberrant. In contrast, the segregation of replication termini was impaired in smc mutant cells. This analysis was extended to germinating spores of an smc mutant. The results showed that during germination, newly duplicated origins, but not termini, were able to separate from each other in the absence of SMC. Also, DAPI (4',6'-diamidino-2-phenylindole) staining revealed that chromosomes in germinating spores were able to undergo partial or complete replication but that the daughter chromosomes were blocked at a late stage in the segregation process. These findings were confirmed by time-lapse microscopy, which showed that after duplication in growing cells the origin regions underwent rapid movement toward opposite poles of the cell in the absence of SMC. This indicates that SMC is not a required component of the mitotic motor that initially drives origins apart after their duplication. It is also concluded that SMC is needed to maintain the proper layout of the chromosome in the cell and that it functions in the cell cycle after origin separation but prior to complete segregation or replication of daughter chromosomes. It is proposed here that chromosome segregation takes place in at least two steps: an SMC-independent step in which origins move apart and a subsequent SMC-dependent step in which newly duplicated chromosomes condense and are thereby drawn apart.     

Chromosome segregation

The ability to visualise specific genes and proteins within bacterial cells is revolutionising knowledge of chromosome segregation. The essential elements appear to be the driving force behind DNA replication, which occurs at fixed cellular positions, the condensation of newly replicated DNA by a chromosome condensation machine located at the cell 1/4 and 3/4 positions, and molecular machines that act at midcell to allow chromosome separation after replication and movement of the sister chromosomes away from the division septum prior to cell division. This review attempts to provide a perspective on current views of the bacterial chromosome segregation mechanism and how it relates to other cellular processes.     

Condensin: crafting the chromosome landscape

The successful transmission of complete genomes from mother to daughter cells during cell divisions requires the structural re-organization of chromosomes into individualized and compact structures that can be segregated by mitotic spindle microtubules. Multi-subunit protein complexes named condensins play a central part in this chromosome condensation process, but the mechanisms behind their actions are still poorly understood. An increasing body of evidence suggests that, in addition to their role in shaping mitotic chromosomes, condensin complexes have also important functions in directing the three-dimensional arrangement of chromatin fibers within the interphase nucleus. To fulfill their different functions in genome organization, the activity of condensin complexes and their localization on chromosomes need to be strictly controlled. In this review article, we outline the regulation of condensin function by phosphorylation and other posttranslational modifications at different stages of the cell cycle. We furthermore discuss how these regulatory mechanisms are used to control condensin binding to specific chromosome domains and present a comprehensive overview of condensin’s interaction partners in these processes.     

The role of DNA replication in chromosome condensation

At metaphase, DNA in a human chromosome is estimated to be compacted at least 10,000 fold in length. However, the higher order mechanisms by which the chromosomes are organized in interphase and subsequently further condensed in mitosis have largely remained elusive. One generally overlooked participant in chromosome condensation is DNA replication. Many early studies of eukaryotic chromosome organization and cell fusions have suggested that DNA replication plays a role in chromosome compaction. Recent phenotypic analysis of Drosophila DNA replication mutants has revitalized this old idea. In this review, the role of DNA replication in chromosome condensation will be examined.     

Non-apoptotic chromosome condensation induced by stress: delineation of interchromosomal spaces

Chromosomes are known to occupy distinct territories, suggesting the existence of definite borders. Visualization of these borders requires chromatin condensation like that seen in prophase cells. We developed a novel method to induce chromosome condensation in all cells regardless of cell cycle stage using a complex set of stresses. The cells were not apoptotic, as indicated by the absence of DNA damage, maintenance of the intact lamina and scaffold attachment factor A, and by the continuation of metabolic processes as well as proliferative capacity. That the appearance of chromosome condensation did not represent a premature mitotic event was shown by the absence of fibrillarin and Ki67 envelopment of chromosomes, continued protein synthesis and the reversibility of chromosome condensation. That chromosome condensation was achieved was demonstrated by the removal of chromatin from the nuclear envelope and chromosome painting. Specific genetic sites known to be at the surface of chromosomes retained their positions as shown by in situ hybridization. Stress-induced chromosome condensation was used to prove that specific nuclear domains such as ND10 are interchromosomally located and that green fluorescent protein-tagged ND10-associated proteins are useful markers for chromosomal boundaries after adenovirus 5 track formation in vivo. From these observations we conclude that chromosomal territories appear to have boundaries that exclude developing macromolecular aggregates. Received: 26 November 1999 / Accepted: 29 February 2000     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

The plant-specific family of DNA-binding proteins containing three HMG-box domains interacts with mitotic and meiotic chromosomes

? The high mobility group (HMG)-box represents a DNA-binding domain that is found in various eukaryotic DNA-interacting proteins. Proteins that contain three copies of the HMG-box domain, termed 3 × HMG-box proteins, appear to be specific to plants. The Arabidopsis genome encodes two 3 × HMG-box proteins that were studied here. ? DNA interactions were examined using electrophoretic mobility shift assays, whereas expression, subcellular localization and chromosome association were mainly analysed by different types of fluorescence microscopy. ? The 3 × HMG-box proteins bind structure specifically to DNA, display DNA bending activity and, in addition to the three HMG-box domains, the basic N-terminal domain contributes to DNA binding. The expression of the two Arabidopsis genes encoding 3 × HMG-box proteins is linked to cell proliferation. In synchronized cells, expression is cell cycle dependent and peaks in cells undergoing mitosis. 3 × HMG-box proteins are excluded from the nuclei of interphase cells and localize to the cytosol, but, during mitosis, they associate with condensed chromosomes. The 3 × HMG-box2 protein generally associates with mitotic chromosomes, while 3 × HMG-box1 is detected specifically at 45S rDNA loci. ? In addition to mitotic chromosomes the 3 × HMG-box proteins associate with meiotic chromosomes, suggesting that they are involved in a general process of chromosome function related to cell division, such as chromosome condensation and/or segregation.     

Relationship between histone phosphorylation and premature chromosome condensation

The relationship between histone phosphorylation and chromosome condensation was investigated by determining changes in phosphorylation levels of histones H1 and H3 following fusion between mitotic and interphase cells and subsequent premature chromosome condensation. We detected significant increases in the levels of phosphorylation of H1 and H3 from interphase chromatin in which a majority of nuclei had undergone premature chromosome condensation. In addition, we noted significant decreases in the phosphate content of the highly phosphorylated mitotic H1 and H3, presumably resulting from phosphatase activity contributed by the interphase component of mitotic/interphase fused cells. These observations further strengthen the correlation between histone phosphorylation and the changes in chromosome condensation associated with the initiation of mitosis. This study also suggests that maintenance of the mitotic chromosomes in a highly condensed state does not require the continued presence of histones in a highly phosphorylated form.     

Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.     

Chromosome segregation in Eubacteria

It is now clear that bacterial chromosomes rapidly separate in a manner independent of cell elongation, suggesting the existence of a mitotic apparatus in bacteria. Recent studies of bacterial cells reveal filamentous structures similar to the eukaryotic cytoskeleton, proteins that mediate polar chromosome anchoring during Bacillus subtilis sporulation, and SMC interacting proteins that are involved in chromosome condensation. A picture is thereby developing of how bacterial chromosomes are organized within the cell, how they are separated following duplication, and how these processes are coordinated with the cell cycle.