Chromosome Separation Research Articles

Isolation and manipulation of meiotic spindles from mouse oocytes reveals migration regulated by pulling force during asymmetric division.

Spindles are essential for accurate chromosome segregation in all eukaryotic cells. This study presents a novel approach for isolating fresh mammalian spindles from mouse oocytes, establishing it as a valuable in vitro model system for a wide range of possible studies. Our method enables the investigation of the physical properties and migration force of meiotic spindles in oocytes. We found that the spindle length decreases upon isolation from the oocyte. Combining this observation with direct measurements of spindle mechanics, we examined the forces governing spindle migration during oocyte asymmetric division. Our findings suggest that the spindle migration is regulated by a pulling force and a net tensile force of approximately 680 pN is applied to the spindle in vivo during the migration process. This method, unveiling insights into spindle dynamics, holds promise as a robust model for future investigations into spindle formation and chromosome separation. We also found that the same approach could not isolate spindles from somatic cells, indicative of mammalian oocytes having a unique spindle organization amenable to isolation.

Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro

Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.

Exploring the role of aggrephagy-related signatures in immune microenvironment, prognosis, and therapeutic strategies of breast cancer.

Breast cancer (BC) is 1 of the most common malignant tumors among women globally. This study aimed to develop a prognostic signature based on aggrephagy-related genes (ARGs). Transcriptomic and clinical data for BC patients were downloaded from the cancer genome atlas and GEO databases. Differential expression analysis, univariate Cox proportional hazards regression and least absolute shrinkage and selection operator Cox regression were employed to construct a prognostic signature. Consensus clustering, evaluation of immune infiltration and drug sensitivity, and gene set enrichment analysis, and development of nomogram were performed. The expression of ARGs was validated using data from the Cancer Cell Line Encyclopedia and clinical samples. Eleven ARGs were abnormally expressed in BC, with 5 showing significant correlations with BC prognosis. Consensus clustering identified 2 molecular subtypes with distinct prognoses. A prognostic signature including 5 ARGs (VIM, TUBB1, TUBA3E, TUBA3D, TUBA1C) was developed, which showed high performance in predicting BC prognosis. The low-risk group showed enrichment in extracellular matrix organization and cell migration processes while chromosome separation was suppressed. Additionally, patients in this group also show activation in several signaling pathways including MAPK, PI3K-AKT, and cAMP pathways, whereas cell cycle and neutrophil extracellular trap formation were significantly inhibited. The signature was also associated with immune infiltration and drug sensitivity. A nomogram incorporating the risk signature, clinical stage and chemotherapy was constructed, demonstrating excellent performance in predicting prognosis. The expression of signature-related genes were validated in patients with BC. This study successfully constructed molecular subtypes and a prognostic signature based on ARGs in BC, and developed a nomogram.

M6A-modified cenRNA stabilizes CENPA to ensure centromere integrity in cancer cells

m6A modification is best known for its critical role in controlling multiple post-transcriptional processes of the mRNAs. Here, we discovered elevated levels of m6A modification on centromeric RNA (cenRNA) in cancerous cells compared with non-cancerous cells. We then identified CENPA, an H3 variant, as an m6A reader of cenRNA. CENPA is localized at centromeres and is essential in preserving centromere integrity and function during mitosis. The m6A-modified cenRNA stabilizes centromeric localization of CENPA in cancer cells during the S phase of the cell cycle. Mutations of CENPA at the Leu61 and the Arg63 or removal of cenRNA m6A modification lead to loss of centromere-bound CENPA during S phase. This in turn results in compromised centromere integrity and abnormal chromosome separation and hinders cancer cell proliferation and tumor growth. Our findings unveil an m6A reading mechanism by CENPA that epigenetically governs centromere integrity in cancer cells, providing potential targets for cancer therapy.

Expression pattern and functional analysis of kinesin-14 KIFC1 in spermatogenesis of Macaca mulatta

C-terminal kinesin motor KIFC1 is increasingly concerned with an essential role in germ cell development. During the spermatogenesis of mice, rats, and crustaceans, KIFC1 functions in regulating meiotic chromosome separation, acrosome vesicle transportation, and nuclear morphology maintenance. The expression pattern of KIFC1 is conservatively concentrated at the acrosome and nucleus of haploid sperm cells. However, whether KIFC1 has similar functions in non-human primates remains unknown. In this study, we constructed the testis-specific cDNA library and cloned different transcripts of KIFC1 based on the genomic sequence. New variants of KIFC1 were identified, and showed different functional domains from the predicted isoforms. The spatio-temporal expression of KIFC1 proteins in seminiferous tubules of rhesus monkeys showed an obvious nuclear localization, specifically expressed in the spermatocytes and early haploid spermatids. The transcripts of KIFC1 also exhibited considerable expression in the nucleus of rhesus LLC-MK2 cells. Besides, we demonstrated that KIFC1 located at the acrosome and microtubule flagella of the mature sperm, and KIFC1 inhibition resulted in sperm tail deformation as well as increased the instability of head-to-tail connection. In summary, this study filled a gap in the reproductive research of the KIFC1 gene in non-human primates.

Revisiting the role of the spindle assembly checkpoint in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae.

Multiple pathways are known to suppress the formation of gross chromosomal rearrangements (GCRs), which can cause human diseases including cancer. In contrast, much less is known about pathways that promote their formation. The spindle assembly checkpoint (SAC), which ensures the proper separation of chromosomes during mitosis, has been reported to promote GCR, possibly by delaying mitosis to allow GCR-inducing DNA repair to occur. Here, we show that this conclusion is the result of an experimental artifact arising from the synthetic lethality caused by the disruption of the SAC and loss of the CIN8 gene, which is often lost in the genetic assay used to select for GCRs. After correcting for this artifact, we find no role of the SAC in promoting GCR.

The central spindle drives anaphase chromosome segregation.

Anaphase chromosome segregation depends on forces exerted by spindle microtubules. In the current model, forces on chromosomes are mediated through the spindle poles: sliding of antiparallel microtubules in the central spindle pushes poles apart, while kinetochore microtubule (kMT) depolymerization pulls chromosomes towards the poles. Here we show that the central spindle is directly linked to the chromosomes rather than the poles in anaphase, based on three lines of evidence. Chromosomes in monopolar spindles can move away from the pole, consistent with forces exerted by antiparallel microtubule sliding. In bipolar spindles, kMT depolymerization is constrained by suppressing central spindle sliding, indicating kinetochore linkage to the central spindle. Finally, increasing the rate of kMT depolymerization slows pole separation without increasing chromosome separation velocity. We conclude that central spindle sliding drives anaphase chromosome separation, while kMT depolymerization limits spindle elongation.

MksB is a novel mycobacterial condensin that orchestrates spatiotemporal positioning of replication machinery

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA.

Aging-associated reduction of chromosomal histones in mammalian oocytes.

Mammalian oocytes undergo a long-term meiotic arrest that can last for almost the entire reproductive lifespan. This arrest occurs after DNA replication and is prolonged with age, which poses a challenge to oocytes in maintaining replication-dependent chromosomal proteins required for the completion of meiosis. In this study, we show that chromosomal histones are reduced with age in mouse oocytes. Both types of histone H3 variants, replication-dependent H3.1/H3.2 and replication-independent H3.3, decrease with age. Aging-associated histone reduction is associated with transcriptomic features that are caused by genetic depletion of histone H3.3. Neither the genetic reduction of chromosomal H3.1/H3.2 nor H3.3 accelerates the aging-associated increase in premature chromosome separation that causes meiotic segregation errors. We suggest that aging-associated reduction of chromosomal histones is linked to several transcriptomic abnormalities but does not significantly contribute to errors in meiotic chromosome segregation during the reproductive lifespan of mice.

Condensin I folds the Caenorhabditis elegans genome.

The structural maintenance of chromosome (SMC) complexes-cohesin and condensins-are crucial for chromosome separation and compaction during cell division. During the interphase, mammalian cohesins additionally fold the genome into loops and domains. Here we show that, in Caenorhabditis elegans, a species with holocentric chromosomes, condensin I is the primary, long-range loop extruder. The loss of condensin I and its X-specific variant, condensin IDC, leads to genome-wide decompaction, chromosome mixing and disappearance of X-specific topologically associating domains, while reinforcing fine-scale epigenomic compartments. In addition, condensin I/IDC inactivation led to the upregulation of X-linked genes and unveiled nuclear bodies grouping together binding sites for the X-targeting loading complex of condensin IDC. C. elegans condensin I/IDC thus uniquely organizes holocentric interphase chromosomes, akin to cohesin in mammals, as well as regulates X-chromosome gene expression.

Live chromosome identifying and tracking reveals size-based spatial pathway of meiotic errors in oocytes.

Meiotic errors of relatively small chromosomes in oocytes result in egg aneuploidies that cause miscarriages and congenital diseases. Unlike somatic cells, which preferentially mis-segregate larger chromosomes, aged oocytes preferentially mis-segregate smaller chromosomes through unclear processes. Here, we provide a comprehensive three-dimensional chromosome identifying-and-tracking dataset throughout meiosis I in live mouse oocytes. This analysis reveals a prometaphase pathway that actively moves smaller chromosomes to the inner region of the metaphase plate. In the inner region, chromosomes are pulled by stronger bipolar microtubule forces, which facilitates premature chromosome separation, a major cause of segregation errors in aged oocytes. This study reveals a spatial pathway that facilitates aneuploidy of small chromosomes preferentially in aged eggs and implicates the role of the M phase in creating a chromosome size-based spatial arrangement.

Overexpression of BubR1 Mitotic Checkpoint Protein Predicts Short Survival and Influences the Progression of Cholangiocarcinoma.

Budding Uninhibited by Benzimidazole-Related 1 (BubR1) or BUB1 Mitotic Checkpoint Serine/Threonine Kinase B (BUB1B) is an essential component of the spindle assembly checkpoint (SAC), which controls chromosome separation during mitosis. Overexpression of BubR1 has been associated with the progression of various cancers. This study demonstrated that high expression of BubR1 correlated with cholangiocarcinogenesis in a hamster cholangiocarcinoma (CCA) model and was associated with shorter survival in patients with CCA. Co-expression of BubR1 and MPS1, which is a SAC-related protein, indicated a shorter survival rate in patients with CCA. Knockdown of BubR1 expression by specific siRNA (siBubR1) significantly decreased cell proliferation and colony formation while inducing apoptosis in CCA cell lines. In addition, suppression of BubR1 inhibited migration and invasion abilities via epithelial-mesenchymal transition (EMT). A combination of siBubR1 and chemotherapeutic drugs showed synergistic effects in CCA cell lines. Taken together, this finding suggested that BubR1 had oncogenic functions, which influenced CCA progression. Suppression of BubR1 might be an alternative option for CCA treatment.

Aurora-B: a novel biomarker inthe invasion and metastasis of osteosarcoma.

Osteosarcoma (OS), a primary human malignant tumor that affects the bones, mostly arises in children and adolescents. Even though surgical resection followed by radiotherapy and chemotherapy has improved the survival rate up to 60%, the long-term positive effect for most patients with OS is not satisfactory. Hence, elucidating the specific mechanisms involved in the pathogenesis of OS is particularly important. Aurora-B, a serine/threonine kinase, plays a crucial role in centrosome regulation, spindle formation and chromosomal separation during mitosis. It has been found that Aurora-B overexpression is related to the occurrence and development of several malignant tumors, including OS. This article summarizes the role of Aurora-B in the invasion and metastasis of OS.

Ssu72 phosphatase deficiency leads to spindle crossing during the second meiotic division process.

Ssu72 is a component of the yeast cleavage/polyadenylation factor (CPF) complex, which catalyzes the dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II at S5-P and S7-P. It has been shown that Ssu72 phosphatase is involved in regulating chromosome cohesion during mitosis. To further clarify whether Ssu72 phosphatase affects chromosome separation during meiotic division in Schizosaccharomyces pombe, we utilized green fluorescent protein (GFP) to label centromeres and red fluorescent protein to label microtubule protein Atb2. The entire meiotic chromosome separation process of ssu72∆ cells was observed in real-time under fluorescence microscope. It was found that two spindles of ssu72∆ cells crossed during the metaphase and anaphase of the second meiotic division, and this spindle crossing led to a new type of spore defect distribution pattern. The results of this study can provide important reference significance for studying the roles of phosphatase Ssu72 in higher organisms.

The cell cycle oscillator and spindle length set the speed of chromosome separation in Drosophila embryos.

Anaphase is tightly controlled in space and time to ensure proper separation of chromosomes. The mitotic spindle, the self-organized microtubule structure driving chromosome segregation, scales in size with the available cytoplasm. Yet, the relationship between spindle size and chromosome movement remains poorly understood. Here, we address how the movement of chromosomes changes during the cleavage divisions of the Drosophila blastoderm. We show that the speed of chromosome separation gradually decreases during the 4 nuclear divisions of the blastoderm. This reduction in speed is accompanied by a similar reduction in the length of the spindle, thus ensuring that these two quantities are tightly linked. Using a combination of genetic and quantitative imaging approaches, we find that two processes contribute to controlling the speed at which chromosomes move at mitotic exit: the activity of molecular motors important for microtubule depolymerization and the cell cycle oscillator. Specifically, we found that the levels of Klp10A, Klp67A, and Klp59C, three kinesin-like proteins important for microtubule depolymerization, contribute to setting the speed of chromosome separation. This observation is supported by quantification of microtubule dynamics indicating that poleward flux rate scales with the length of the spindle. Perturbations of the cell cycle oscillator using heterozygous mutants of mitotic kinases and phosphatases revealed that the duration of anaphase increases during the blastoderm cycles and is the major regulator of chromosome velocity. Thus, our work suggests a potential link between the biochemical rate of mitotic exit and the forces exerted by the spindle. Collectively, we propose that the cell cycle oscillator and spindle length set the speed of chromosome separation in anaphase.

A haplotype-resolved reference genome of a long-distance migratory bat, Pipistrellus nathusii (Keyserling & Blasius, 1839).

We present a complete, chromosome-scale reference genome for the long-distance migratory bat Pipistrellus nathusii. The genome encompasses both haplotypic sets of autosomes and the separation of both sex chromosomes by utilizing highly accurate long-reads and preserving long-range phasing information through the use of three-dimensional chromatin conformation capture sequencing (Hi-C). This genome, accompanied by a comprehensive protein-coding sequence annotation, provides a valuable genomic resource for future investigations into the genomic bases of long-distance migratory flight in bats as well as uncovering the genetic architecture, population structure and evolutionary history of Pipistrellus nathusii. The reference-quality genome presented here gives a fundamental resource to further our understanding of bat genetics and evolution, adding to the growing number of high-quality genetic resources in this field. Here, we demonstrate its use in the phylogenetic reconstruction of the order Chiroptera, and in particular, we present the resources to allow detailed investigations into the genetic drivers and adaptations related to long-distance migration.

Unfurling an improved method for visualizing mitotic chromosomes in ferns.

Cytotaxonomy employs chromosome visualization to study organismal relationships and evolution. Despite the critical value of cytogenetic data, cytotypes are lacking for many plant groups. Here, we present an improved approach for visualizing mitotic chromosomes in ferns, a key lineage of land plants, using the dividing cells of unfurling croziers (fiddleheads). Our modified mitotic chromosome preparation incorporates a brief pectinase-cellulase pretreatment, as well as colchicine fixation and the Feulgen reaction to improve the staining and separation of mitotic chromosomes. To demonstrate this easy and efficient assessment, we determined the sporophytic (2n) chromosome number for three fern species: Cheilanthes mollis (2n = 60), Cheilanthes hypoleuca (2n = 120), and Nephrolepis cordifolia (2n = 82). The new method presented here improves visualizations of mitotic chromosomes from the dividing nuclei of young fern croziers. Fiddleheads are widely accessible in nature and in living collections worldwide, and this modified approach increases their suitability for fern cytotaxonomic studies.

Effect of Treatments of Colchicine, Diethyl Sulphate and Triethylamine in Tomato (<I>Lycopersicon esculentum</I> Mill.)

The present investigation was undertaken in one variety of Tomato, Best of All, to compare the cytogenetic effects of treatments of seeds of tomato with colchicine, diethyl sulphate and triethylamine and also to compare the morphological and physiological effects of these chemicals.Lowest number of seeds germinated from diethyl sulphate treatment. Plants originating from colchicine, diethyl sulphate and triethylamine treatments showed many variant types. Interesting among these were those showing spotting on leaves originating from diethyl sulphate treatment, dwarf ones originating from colchicine and diethyl sulphate treatments and those with abnormal leaves originating from treatment with all the three chemicals.Mitotic irregularities included clumped chromosomes and fragments at metaphase and bridges and fragments at anaphase while in meiosis clumped chromosomes, univalents, trivalents and quadrivalents were noted at metaphase I. Anaphase I irregularities included cells with fragments, bridges with or without fragments, lagging chromosomes and unequal separation of chromosomes. In metaphase II cells with aneuploid chromosome numbers and those with fragments were noticed while in anaphase II cells were found with fragments and unequal separation of chromosomes.The importance of detecting interesting mutants in the Ma generation and the importance of finding translocations and inversions have been pointed out.

Divergent selection for feed efficiency in pigs altered the duodenum transcriptomic response to feed intake and its DNA methylation profiles.

Feed efficiency is a trait of interest in pigs as it contributes to lowering the ecological and economical costs of pig production. A divergent genetic selection experiment from a Large White pig population was performed for 10 generations, leading to pig lines with relatively low- (LRFI) and high- (HRFI) residual feed intake (RFI). Feeding behavior and metabolic differences have been previously reported between the two lines. We hypothesized that part of these differences could be related to differential sensing and absorption of nutrients in the proximal intestine. We investigated the duodenum transcriptome and DNA methylation profiles comparing overnight fasting with ad libitum feeding in LRFI and HRFI pigs (n = 24). We identified 1,106 differentially expressed genes between the two lines, notably affecting pathways of the transmembrane transport activity and related to mitosis or chromosome separation. The LRFI line showed a greater transcriptomic response to feed intake than the HRFI line. Feed intake affected genes from both anabolic and catabolic pathways in the pig duodenum, such as rRNA production and autophagy. Several nutrient transporter and tight junction genes were differentially expressed between lines and/or by short-term feed intake. We also identified 409 differentially methylated regions in the duodenum mucosa between the two lines, while this epigenetic mark was less affected by feeding. Our findings highlighted that the genetic selection for feed efficiency in pigs changed the transcriptome profiles of the duodenum, and notably its response to feed intake, suggesting key roles for this proximal gut segment in mechanisms underlying feed efficiency.NEW & NOTEWORTHY The duodenum is a key organ for the hunger/satiety loop and nutrient sensing. We investigated how the duodenum transcriptome and DNA methylation profiles are affected by feed intakes in pigs. We observed thousands of changes in gene expression levels between overnight-fasted and fed pigs in high-feed efficiency pig lines, but almost none in the related low-feed efficiency pig line.

C. elegans spermatocyte divisions show a weak spindle checkpoint response.

Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies have shown that spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in Caenorhabditis elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillates between the two divisions. However, the SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.