Showing papers on "Metaphase published in 2006"
TL;DR: In mammalian cells, it is found that chromosomes can congress before becoming bioriented, and this congression mechanism depended on the kinetochore-associated, plus end–directed microtubule motor CENP-E (kinesin-7).
Abstract: The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).
438 citations
TL;DR: The results support a preanaphase correction mechanism for merotelic attachments in which correct plus-end attachments are pulled away from high concentrations of Aurora B at the inner centromere, and incorrect merotELic attachments are destabilized by being pulled toward the inner Centromere.
421 citations
TL;DR: Improved methods for obtaining, preparing, and staining fish chromosomes are described and procedures for resolving serial or G-type bands are presented.
Abstract: Improved methods for obtaining, preparing, and staining fish chromosomes are described. Included are procedures for resolving serial or G-type bands. A brief review of various metaphase banding procedures and their use in fishes is also presented.
271 citations
TL;DR: In this paper, the authors reported 5 cases (World Health Organization grade III) in which metaphase cytogenetics identified a derivative chromosome consisting of what appears to be the whole arms of 1q and 19p forming a der(1;19)(q10;p10).
Abstract: Deletions of portions of chromosomes 1p and 19q are closely associated with the oligodendroglioma histologic phenotype. In most cases, 1p and 19q are codeleted, yet the mechanism of dual loss is unexplained. We report 5 cases (World Health Organization grade III) in which metaphase cytogenetics identified a derivative chromosome consisting of what appears to be the whole arms of 1q and 19p forming a der(1;19)(q10;p10). Metaphase fluorescent in situ hybridization (FISH) confirmed the derivative chromosome was composed of 1q and 19p material in 3 cases; in 2 cases with few metaphases, FISH confirmed 19p material on the derivative chromosome. In all cases, interphase FISH showed net loss of 1p and 19q in 77% to 92% of cells, and microsatellite studies were consistent with 1p and 19q loss. We hypothesize the following: occurrence of a balanced whole-arm translocation between chromosomes 1 and 19 forming 2 derivative chromosomes, one composed of 1q and 19p, the other of 1p and 19q. Subsequent loss of the der(1;19)(p10;q10) then results in the simultaneous 1p and 19q loss observed in oligodendroglioma with retention of the der(1;19)(q10;p10) seen in these cases.
271 citations
TL;DR: It is shown that PP2A(Cdc55) phosphatase keeps Net1 underphosphorylated in metaphase, facilitating Cdk-dependent Net1 phosphorylation and presenting a new quantitative model for mitotic exit in budding yeast.
265 citations
TL;DR: The identification and characterisation of a novel spindle and kinetochore (KT)‐associated complex that is required for timely anaphase onset and the data suggest that the Ska1/2 complex plays a critical role in the maintenance of the metaphase plate and/or spindle checkpoint silencing.
Abstract: Chromosome segregation during mitosis requires chromosomes to undergo bipolar attachment on spindle microtubules (MTs) and subsequent silencing of the spindle checkpoint Here, we describe the identification and characterisation of a novel spindle and kinetochore (KT)-associated complex that is required for timely anaphase onset The complex comprises at least two proteins, termed Ska1 (Spindle and KT Associated 1) and Ska2 Ska1 associates with KTs following MT attachment during prometaphase Ska1 and Ska2 interact with each other and Ska1 is required for Ska2 stability in vivo Depletion of either Ska1 or Ska2 by small interfering RNA results in the loss of both proteins from the KT The absence of Ska proteins does not disrupt overall KT structure, but KT fibres show an increased cold-sensitivity Most strikingly, Ska-depleted cells undergo a prolonged checkpoint-dependent delay in a metaphase-like state This delay is characterised by the recruitment of Mad2 protein to a few KTs and the occasional loss of individual chromosomes from the metaphase plate These data suggest that the Ska1/2 complex plays a critical role in the maintenance of the metaphase plate and/or spindle checkpoint silencing
234 citations
TL;DR: By expressing Cre recombinase from a zona pellucida promoter, a floxed allele of separase is deleted and prevents removal of Rec8 from chromosome arms and resolution of chiasmata, which hinders extrusion of the first polar body (PBE) and causes female sterility.
231 citations
TL;DR: It is demonstrated that FISH analysis underestimates the complexity of chromosomal aberrations in CLL, therefore, conventional cytogenetics may define subgroups of patients with high risk of progression.
212 citations
TL;DR: Analysis of seven ICEN components with unknown function suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP‐H and hMis6 onto the centromere.
Abstract: The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.
196 citations
TL;DR: In this article, it was shown that INCENP phosphorylation by cyclin-dependent kinase 1 (Cdk1) is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of plk1 and Aurora-B may play crucial roles in the regulation of chromosomal dynamics.
Abstract: Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.
189 citations
TL;DR: It is proposed that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis.
Abstract: The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood In this paper, we analyze the functions of six kinetochore-bound MT-associated proteins (kMAPs) using RNAi, live-cell microscopy and quantitative image analysis We find that RNAi-mediated depletion of two kMAPs, the adenomatous polyposis coli protein (APC) and its binding partner, EB1, are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore–MT attachment per se Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation We propose that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis
TL;DR: In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold and the combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of amacromolecular interaction, all of which are important for modeling protein interaction networks.
TL;DR: It is concluded that normal metaphase spindle organisation and the transition to anaphase is dependent on inactivation of AtMAP65-1, and that CDK and MAPK are involved in this phosphorylation.
Abstract: Cell division depends on the fine control of both microtubule dynamics and microtubule organisation. The microtubule bundling protein MAP65 is a `midzone MAP9 essential for the integrity of the anaphase spindle and cell division. Arabidopsis thaliana MAP65-1 (AtMAP65-1) binds and bundles microtubules by forming 25 nm cross-bridges. Moreover, as AtMAP65-1 bundles microtubules in interphase, anaphase and telophase but does not bind microtubules in prophase or metaphase, its activity through the cell cycle must be under tight control. Here we show that AtMAP65-1 is hyperphosphorylated during prometaphase and metaphase and that CDK and MAPK are involved in this phosphorylation. This phosphorylation inhibits AtMAP65-1 activity. Expression of non-phosphorylatable AtMAP65-1 has a negative effect on mitotic progression resulting in excessive accumulation of microtubules in the metaphase spindle midzone causing a delay in mitosis. We conclude that normal metaphase spindle organisation and the transition to anaphase is dependent on inactivation of AtMAP65-1.
TL;DR: A Brd4-Tat fusion protein that is efficiently taken up by different transformed cells harboring HPV plasmids is developed, suggesting that such peptides may lead to the development of inhibitors of latency for many, if not all, papillomaviruses.
TL;DR: Roles for structurally diverse motors in the complex processes of chromosome segregation are demonstrated and important similarities and intriguing differences between higher and lower eukaryotes are revealed.
Abstract: Accurate chromosome segregation during mitosis requires biorientation of sister chromatids on the microtubules (MT) of the mitotic spindle. Chromosome–MT binding is mediated by kinetochores, which are multiprotein structures that assemble on centromeric (CEN) DNA. The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor proteins at yeast kinetochores have yet to be determined, despite evidence of their importance in higher eukaryotes. We show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores and function in three distinct processes. Kip1p and Cin8p, which are kinesin-5/BimC family members, cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a kinesin-8,-13/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The kinesin-14 motor Kar3p appears to function at the subset of kinetochores that become detached from spindle MTs. These data demonstrate roles for structurally diverse motors in the complex processes of chromosome segregation and reveal important similarities and intriguing differences between higher and lower eukaryotes.
TL;DR: A force-balance model is developed that describes Drosophila embryo chromosome motility in terms of a balance of forces acting on kinetochores and kMTs that is generated by multiple polymer ratchets and mitotic motors coupled to tension-dependent kMT dynamics.
TL;DR: It is demonstrated that this process is required for the phosphorylation of MKlp1 at S911, an Aurora B consensus site overlapping a bipartite nuclear localization sequence (NLS).
TL;DR: Assessment of the localization of 13 different animal and human E2 proteins from seven papillomavirus genera shows that most of them are stably bound to chromosomes throughout mitosis, and there are variations in the specificity of this binding.
Abstract: The E2 protein segregates episomal bovine papillomavirus (BPV) genomes to daughter cells by tethering them to mitotic chromosomes, thus ensuring equal distribution and retention of viral DNA. To date, only the BPV1 E2 protein has been shown to bind to mitotic chromosomes. We assessed the localization of 13 different animal and human E2 proteins from seven papillomavirus genera, and we show that most of them are stably bound to chromosomes throughout mitosis. Furthermore, in contrast to the random association of BPV1 E2 with mitotic chromosomes, several of these proteins appear to bind to more specific regions of mitotic chromosomes. Using human papillomavirus (HPV) type 8 E2, we show that this region is adjacent to centromeres/kinetochores. Therefore, E2 proteins from both HPV and animal papillomavirus bind to mitotic chromosomes, and there are variations in the specificity of this binding. Only the α-papillomavirus E2 proteins do not stably associate with mitotic chromatin throughout mitosis. These proteins closely associate with prophase chromosomes and bind to chromosomes in telophase but not in metaphase. However, extraction of mitotic cells before fixation results in α-E2 proteins binding to the pericentromeric region of metaphase chromosomes, as observed for HPV8 E2. We postulate that this is the authentic target of these E2 proteins but that additional factors or a specialized cellular environment is required to stabilize this association. Thus, E2-mediated tethering of viral genomes to mitotic chromosomes is a common strategy of papillomaviruses, but different viruses have evolved different variations of this theme.
TL;DR: Every mitosis, replicated chromosomes must be accurately segregated into each daughter cell, so that sisters separate and are pulled to opposite poles during anaphase.
Abstract: Every mitosis, replicated chromosomes must be accurately segregated into each daughter cell. Pairs of sister chromatids attach to the bipolar mitotic spindle during prometaphase, they are aligned at metaphase, then sisters separate and are pulled to opposite poles during anaphase. Failure to attach
TL;DR: It is shown that cyclinB1 is able to maintain a bipolar spindle even after sister chromatids had become separated and suggest an important role of hKid in this process.
Abstract: The disassembly of the mitotic spindle and exit from mitosis require the inactivation of Cdk1. Here, we show that expression of nondegradable cyclinB1 causes dosedependent mitotic arrest phenotypes. By monitoring chromosomes in living cells, we determined that pronounced overexpression of stable cyclinB1 entailed metaphase arrest without detectable sister chromatid separation, while moderate overexpression arrested cells in a pseudometaphase state, in which separated sister chromatids were kept at the cellular equator by a bipolar ‘metaphase-like’ spindle. Chromosomes that left the pseudometaphase plate became pulled back and individual kinetochores were found to be merotelically attached to both spindle poles in stable cyclinB1 arrested cells. Inactivation of the chromokinesin hKid, by RNAi or antibody microinjection, prevented the formation of stable bipolar spindles and the ‘metaphase-like’ alignment of chromosomes in cells expressing stable cyclinB1. These experiments show that cyclinB1 is able to maintain a bipolar spindle even after sister chromatids had become separated and suggest an important role of hKid in this process. Cells expressing low levels of nondegradable cyclinB1 progressed further in mitosis and arrested in telophase.
TL;DR: Studies on Erp1/Emi2 regulation have led to a detailed molecular understanding of the Ca2+-mediated release from CSF arrest that occurs upon fertilization.
Abstract: Fertilization is the fundamental process in which two gametes - sperm and oocyte - fuse to generate a zygote that will form a new multicellular organism. In most vertebrates, oocytes await fertilization while arrested at metaphase of meiosis II. This resting state can be stable for many hours and depends on a cytoplasmic activity termed cytostatic factor (CSF). Recently, members of the novel Emi/Erp family of proteins have been put forward as important components of CSF. These proteins inhibit the anaphase-promoting complex/cyclosome (APC/C), which acts at the very core of the cell cycle regulatory machinery. Initially, Xenopus early mitotic inhibitor 1 (Emi1) was proposed to be a component of CSF, but newer work suggests that a structural relative, Emi-related protein 1 (Erp1/Emi2), is essential for maintenance of CSF arrest in Xenopus. Most importantly, studies on Erp1/Emi2 regulation have led to a detailed molecular understanding of the Ca2+-mediated release from CSF arrest that occurs upon fertilization.
TL;DR: Multiple meiotic genes are aberrantly activated during mitotic catastrophe in p53 mutated lymphoma cells after irradiation and it is suggested that the coordinated expression of MOS and REC8 regulate the extent of arrested mitoses and polyploidy.
Abstract: We have previously reported that p53 mutated radioresistant lymphoma cell lines undergo mitotic catastrophe after irradiation, resulting in metaphase arrest and the generation of endopolyploid cells. A proportion of these endopolyploid cells then undergo a process of de-polyploidisation, stages of which are partially reminiscent of meiotic prophase. Furthermore, expression of meiosis-specific proteins of the cancer/testis antigens group of genes has previously been reported in tumours. We therefore investigated whether expression of meiosis-specific genes was associated with the polyploidy response in our tumour model. Three lymphoma cell lines, Namalwa, WI-L2-NS and TK6, of varying p53 status were exposed to a single 10 Gy dose of gamma radiation and their responses assessed over an extended time course. DNA flow cytometry and mitotic counts were used to assess the kinetics and extent of polyploidisation and mitotic progression. Expression of meiotic genes was analysed using RT-PCR and western blotting. In addition, localisation of the meiotic cohesin REC8 and its relation to centromeres was analysed by immunofluorescence. The principal meiotic regulator MOS was found to be significantly post-transcriptionally up-regulated after irradiation in p53 mutated but not p53 wild-type lymphoma cells. The maximum expression of MOS coincided with the maximal fraction of metaphase arrested cells and was directly proportional to both the extent of the arrest and the number of endopolyploid cells that subsequently emerged. The meiotic cohesin REC8 was also found to be up-regulated after irradiation, linking sister chromatid centromeres in the metaphase-arrested and subsequent giant cells. Finally, RT-PCR revealed expression of the meiosis-prophase genes, DMC1, STAG3, SYCP3 and SYCP1. We conclude that multiple meiotic genes are aberrantly activated during mitotic catastrophe in p53 mutated lymphoma cells after irradiation. Furthermore, we suggest that the coordinated expression of MOS and REC8 regulate the extent of arrested mitoses and polyploidy.
TL;DR: Because of the biochemically accessible nature of the egg extract system, a wide array of biochemical techniques can be combined with spindle and chromosome assembly reactions to evaluate the roles of specific proteins in these processes.
Abstract: Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and "cycling" of the reaction through interphase and back into metaphase promotes formation of bipolar spindles capable of segregating their duplicated chromosomes. Reactions can be "spun down" onto cover slips for immunofluorescence analysis. High-speed extracts support mitotic chromosome condensation, which can be observed live by fluorescence time-lapse video microscopy. Because of the biochemically accessible nature of the egg extract system, a wide array of biochemical techniques can be combined with spindle and chromosome assembly reactions to evaluate the roles of specific proteins in these processes.
TL;DR: It is found that efficient plus-end coupling is not required for maintenance of chromosome biorientation, maintenance of tension between sister kinetochores, or (3) chromosome segregation.
TL;DR: It is found that CLIP‐170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules, and facilitates the formation of Kinetochore–microtubule attachments, possibly through direct capture of microtubule at the kinetchore.
Abstract: CLIP-170 is a microtubule ‘plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore–microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore–microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore–microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore–microtubule attachments, possibly through direct capture of microtubules at the kinetochore.
TL;DR: It is demonstrated that 8-oxoG is unevenly distributed in the normal human genome and that the distribution pattern is conserved among different individuals, and regions with a high frequency of recombination and single nucleotide polymorphisms (SNPs) are preferentially located within chromosomal regions withA high density of 8-OxoG.
Abstract: 8-Oxoguanine (8-oxoG), a major spontaneous form of oxidative DNA damage, is considered to be a natural cause of genomic diversity in organisms because of its mutagenic potential. The steady-state level of 8-oxoG in the nuclear genome of a human cell has been estimated to be several residues per 10(6) guanines. In the present study, to clarify the genome-wide distribution of 8-oxoG in the steady state, we performed fluorescence in situ detection of 8-oxoG on human metaphase chromosomes using a monoclonal antibody. Multiple dot-like signals were observed on each metaphase chromosome. We then mapped the position of the signal at megabase resolution referring to the cytogenetically identified chromosomal band, and demonstrated that 8-oxoG is unevenly distributed in the normal human genome and that the distribution pattern is conserved among different individuals. Moreover, we found that regions with a high frequency of recombination and single nucleotide polymorphisms (SNPs) are preferentially located within chromosomal regions with a high density of 8-oxoG. Our findings suggest that 8-oxoG is one of the main causes of frequent recombinations and SNPs in the human genome, which largely contribute to the genomic diversity in human beings.
TL;DR: The results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined, and it is suggested that “residual” foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.
Abstract: Suzuki, M., Suzuki, K., Kodama, S. and Watanabe M. Phosphorylated Histone H2AX Foci Persist on Rejoined Mitotic Chromosomes in Normal Human Diploid Cells Exposed to Ionizing Radiation. Radiat. Res. 165, 269–276 (2006). Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal brid...
TL;DR: The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition and early exit from mitosis after chromosome segregation.
Abstract: Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition.
TL;DR: The structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40–50 nm thick.
Abstract: Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40–50 nm thick. On average, each S2 kinetochore binds 11±2 MTs, in contrast to the 4–6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.
TL;DR: A model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles is proposed.
Abstract: Anastral meiotic spindles are thought to be organized differently from astral mitotic spindles, but the field lacks the basic structural information required to describe and model them, including the location of microtubule-nucleating sites and minus ends. We measured the distributions of oriented microtubules in metaphase anastral spindles in Xenopus laevis extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We found that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. Based on these data, we propose a model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles.