Bcl-2 is an integral component of mitotic chromosomes

We previously demonstrated that phospho-Thr56 Bcl-2 colocalizes with Ki-67 and nucleolin in nuclear structures in prophase cells and is detected on mitotic chromosomes in later mitotic phases. To gain insight into the fine localization of Bcl-2 on mitotic chromosomes, we further investigated Bcl-2 localization by immunostaining of Bcl-2 with known components of metaphase chromosomes and electron microscopic immunocytochemistry. Immunofluorescence analysis on HeLa mitotic cells together with chromatin immunoprecipitation assays showed that Bcl-2 is associated with the condensed chromatin. Co-immunostaining experiments performed on mitotic chromosome spreads demonstrated that Bcl-2 is not localized on the longitudinal axis of chromatids with the condensin complex, but partially colocalizes with histone H3 on some regions of the mitotic chromosome. Finally, most of the Bcl-2 staining overlaps with Ki-67 staining at the chromosome periphery. Bcl-2 localization at the periphery and over the mitotic chromosome was confirmed by immunoelectron microscopy on mitotic cells.Our results indicate that Bcl-2 is an integral component of the mitotic chromosome.     

Immunofluorescent localisation of cytokeratin antigens in mitotic HeLa cells using monoclonal antibodies

The organisation of cytokeratin filaments in mitotic HeLa cells has been analysed by immunofluorescence microscopy using a monoclonal antibody which recognises proteins with apparent subunit molecular weights of 52 kDa and 57 kDa and which binds exclusively to cytokeratin-type filaments. Mitotic cells were prepared for microscopic analysis by hypotonic swelling, centrifugation onto glass slides, brief pre-extraction with 0.1% Triton X-100 and fixation in 80% ethanol. This procedure gave particularly good resolution of intermediate filaments and preservation of chromosome morphology. In prometaphase-metaphase cells the antigen was present in an anastomosing filament network which completely or partially enclosed the chromosomes, in filament fragments and in cytoplasmic aggregates. The epichromosomal filament network was absent from cells in anaphase or later stages of mitosis. In these cells non-filamentous antigen was often located in a narrow band defining the periphery of individual chromosomes and in variable numbers of cytoplasmic filaments or fragments. The results suggest that extensive disaggregation and reformation of cytokeratin filaments occurs during mitosis and that disaggregated cytokeratin proteins are frequently located adjacent to mitotic chromosomes.     

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.     

Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G1 period of the HeLa cell cycle. HeLa cells synchronized in the G1 period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N2O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G1 period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G1 period, when DNA synthesis is initiated.     

The mitotic chromosome is an assembly of rigid elastic axes organized by structural maintenance of chromosomes (SMC) proteins and surrounded by a soft chromatin envelope

The structure of mitotic chromosomes is still poorly understood. Here we describe the use of a novel approach based on elasticity measurements of a single chromosome for studying the organization of these objects. The data reveal that mitotic chromosomes exhibit a non-homogenous structure consisting of rigid elastic axes surrounded by a soft chromatin envelope. The chemical continuity of DNA, but not RNA, was required for the maintenance of these axes. The axes show a modular structure, and the structural maintenance of chromosomes (SMC) proteins participate in their organization. Topoisomerase II was not involved in either the organization of the axes or the maintenance of the mitotic chromosomes. A model for the assembly and the structure of the mitotic chromosome is proposed. According this model, the chromosome axes are dynamic structures that assemble at the onset and disassemble the end of mitosis, respectively. The SMC proteins, in addition to maintaining axis elasticity, are essential for the determination of the rod-like chromosome shape. The extreme compaction of mitotic chromosomes is determined mainly by the high amount of bivalent ions bound to DNA at mitosis.     

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.     

Immunolocalization of Ku-proteins (p80/p70): Localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes

Distribution on both nuclei and metaphase chromosomes of Ku-proteins, recognized by autoantibodies from a patient with systemic lupus erythematosus, has been studied using a specific monoclonal antibody (mAbH6) that recognizes p70, one Ku-protein. Observation with either a conventional fluorescent microscope or a confocal laser scanning microscope revealed mAbH6-stained p70 antigen localized on both nuclear periphery and nucleoli of human interphase cells. The specific staining of nucleoli with mAbH6 has been confirmed using isolated nucleoli from rat liver in which the staining was seen as fine granules surrounding nucleolar DNA. During mitosis p70 antigen moved away from association with the nuclear envelope region to localization on the periphery of condensed chromosomes with no apparent staining of chromosome interior. The p70 antigen was copurified with DNA fragments by immunoaffinity column chromatography using mAbH6. The mAbH6 staining of both nuclear periphery and nucleoli was lost upon digestion with DNase I at low concentrations. These results suggest that p70 antigen is connected with these nuclear structures through DNA.     

The SUMO-targeted ubiquitin ligase RNF4 localizes to etoposide-exposed mitotic chromosomes: Implication for a novel DNA damage response during mitosis

RNF4, a SUMO-targeted ubiquitin ligase (STUbL), localizes to the nucleus and functions in the DNA damage response during interphase of the cell cycle. RNF4 also exists in cells undergoing mitosis, where its regulation and function remain poorly understood. Here we showed that administration of etoposide, an anticancer DNA topoisomerase II poison, to mitotic human cervical cancer HeLa cells induced SUMO-2/3-dependent localization of RNF4 to chromosomes. The FK2 antibody signals, indicative of poly/multi-ubiquitin assembly, were detected on etoposide-exposed mitotic chromosomes, whereas the signals were negligible in cells depleted for RNF4 by RNA interference. This suggests that RNF4 functions as a STUbL in the etoposide-induced damage response during mitosis. Indeed, RNF4-depletion sensitized mitotic HeLa cells to etoposide and increased cells with micronuclei. These results indicate the importance of the RNF4-mediated STUbL pathway during mitosis for the maintenance of chromosome integrity and further implicate RNF4 as a target for topo II poison-based therapy for cancer patients.     

Stability in association of the peripheral material with mitotic chromosomes

The localization of nucleolar proteins (fibrillarin and B-23), and of the protein of interphase nuclear matrix (NMP-65) was studied in the perichromosomal material (CM) after of short hypotonic treatment (15% solution of Henks medium) on cultured pig embryonic kidney cells, followed by restoration of isotonic conditions. It is shown that during hypotonic shock the mitotic chromosomes demonstrate reversible swelling, but their periphery is bounded with a rim of PCM, containing antibodies to fibrillarin and NMP-65, but not to B-23. After returning the cells to the initial isotonic medium, all the three proteins can be detected again on the periphery of chromosomes. It suggests the existence of different stability in the association of free proteins with chromosome bodies. Besides, B-23 and fibrillarin could be visualized in residual nucleoli after a complete extraction of histones and DNA from nuclei.     

Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.     

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.     

Nuclear matrix proteins (with molecular masses of 38 and 50 kDa) are transported by chromosomes in mitosis

Using the immunofluorescence method, sera M-68 and K-43 from patients with autoimmune diseases were shown to stain interphase nuclei and the periphery of mitotic chromosomes of pig embryo kidney cells. Western blotting revealed a polypeptide with a molecular mass of 50 kDa in M-68 serum and polypeptide with a molecular mass 38 kDa in K-43 serum. In the nuclear protein matrix, the antibodies to protein with a molecular mass of 38 kDa stained only the nucleolar periphery, while the antibodies to protein with a molecular mass of 50 kDa stained not only the nucleolar periphery, but also all interphase nuclei. It was shown that, among all components of the nuclear protein matrix (lamina, internuclear network, residual nucleoli), only the nucleolar periphery contained the 38-kDa protein, while the 50-kDa protein was part of the residual nucleolar periphery and participated in the formation of a nuclear-protein network. Both proteins in interphase cell in situ were located in nuclei, but one of them with a molecular mass of 50 kDa was in the form of small, clearly outlined granules, while the other protein (38 kDa) was in the form of small, bright granules on a background of a diffusely stained nucleus. Both proteins also were revealed as a continuous rim around the nucleolar periphery. During all mitotic stages, the 50-kDa protein was seen over the whole chromosomal periphery as a sheath, while the 38-kDa protein formed individual fragments and granules around them. After the decondensation of the nucleus and chromosomes induced by hypotonic treatment, both antibodies stained interphase nuclei diffusely, whereas, in mitotic cells, they stained the surfaces of swollen chromosomes. Polypeptide with a molecular mass of 50 kDa maintained a strong connection with the periphery of the chromosome in the norm during decondensation induced by hypotonic treatment and during subsequent recondensation in isotonic medium, while, during recondensation, protein with a molecular mass of 38 kDa partially lost contact with the chromosome and, at the same time, appeared in the form of granules in the cytoplasm. The obtained data allow one to conclude that nuclear matrix proteins can be transferred with peripheral chromosomal material; similar to the main nucleolar proteins (fibrillarin, B-23, nucleolin, et al.) and some non-nucleolar components of the nuclear protein matrix, they can also have connections of different stabilities with chromosomal periphery.     

Location of nuclear antigen(s) recognized by DSB389 MAb, a monoclonal antibody against desmin, observed by confocal laser scanning fluorescence microscopy

The location of antigen(s) of DSB389 MAb, a monoclonal antibody which was raised against an intermediate filament protein, desmin, was investigated in HeLa S3 cells by indirect immuno-electron microscopy and by confocal laser scanning fluorescence microscopy. At interphase, the antigen(s) locates mainly in the intra-nuclear space adjacent to chromatin and sometimes near the nuclear periphery. At mitosis, they locate at the periphery of the chromosomes -first at each chromosome, then around the mass of chromosomes. The antigen(s) are thought to be a component of one type of nuclear matrix which is present outside both chromatin and chromosomes and which has some structural role(s) in organizing them in the nucleus or in the nuclear region.     

Ultrastructural composition of nonhistone chromosomal skeleton at different stages of mitosis in situ

In interphase cells of the SPEV culture treated with Triton X-100, 2 M NaCl, and DNAse, in the presence of 2 mM CuCl₂, we clearly revealed a stabilized nuclear protein material (NPM) composed of a peripheral lamina, residual nucleolus, and internal fibrillar network. This network is formed by thin fibrils 10–20 nm in diameter, which are also revealed in the nonhistone matrix of mitotic chromosomes at all stages of mitosis. In mitotic chromosomes, NPM is represented as a network of the 10–20-nm-thick fibrils without any features of the central-axial structures. Beginning from the middle prophase, it is possible to see approached sister chromatids in contact with each other in certain sites, similar to centromeres. At these sites, the thickness of fibrils increases up to 40–50 nm, whereas the fibrils themselves are disposed more tightly; this structure can be seen in the chromosome until telophase. At the end of telophase, the decondensation of chromosomes and formation of two new nuclei whose NPM is analogous to NPM of usual interphase nucleus are observed. Thus, the NPM elements can perform the role of a skeleton in both the interphase nucleus and mitotic chromosomes.     

Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.