Chromosome Alignment Research Articles

Efficacy of plogosertib (CYC149462), a novel orally bioavailable PLK1 inhibitor, on patient-derived models of colorectal cancer.

209 Background: Colorectal cancer (CRC) is the third most common cancer in adults in the United States. Given the relative rapid cell division in cancer cells compared to most normal cells, drugs targeting mitosis and other cell cycle checkpoints are of interest in CRC treatment. One multi-tasking protein in cell division is polo-like kinase 1 (plk1). Plk1 is a serine threonine kinase that is highly expressed during mitosis and involved in a variety of functions, including spindle assembly, chromosome segregation, G2/M transition, and cytokinesis. Encouragingly, its expression is also significantly higher in CRC compared to normal mucosa, which makes it an attractive cancer-specific target in CRC. In this study, we sought to evaluate the efficacy of plogosertib (CYC149462), a novel plk1 inhibitor, in a series of preclinical models of advanced CRC. Methods: Fourteen CRC patient-derived organoids (PDOs) were generated from patients who underwent biopsy or resection for their primary or metastatic CRC under an IRB approved protocol at Duke University. Diagnosis of CRC is confirmed in PDOs using H&E and IHC. Subsequently, PDOs were treated with plogosertib in a range of concentrations from 256pM to 100µM. Drug screens were performed for standard of care agents 5FU, oxaliplatin, and SN38. Viability was assessed with Cell-Titer Glo (CTG) 72 hours after treatment. Using a matched PDX, mice were treated with vehicle or 40 mg/kg daily of plogosertib via oral gavage for 2 weeks, 5 days per week. Cell cycle analysis was performed by flow cytometry, and localization of DNA during mitosis was done by fluorescence staining. Results: CRC PDOs were more sensitive to plogosertib (CYC149462) than 5FU, and oxaliplatin, with significantly lower IC 50 values (518.86±377.47nM for plogosertib vs. 38.87±45.63µM for 5FU, and 37.78±39.61µM for oxaliplatin respectively (ANOVA, P < 0.05). Subsequently, we validated our findings in vivo using a matched PDX in which plogosertib treatment led to significant tumor growth inhibition compared to vehicle (t-test, p < 0.05) without serious adverse effects. Furthermore, we showed that plogosertib induces dysfunction in alignment of chromosomes and subsequent G2/M cell cycle arrest (p < 0.05). Conclusions: Together, our study shows that pharmacological inhibition of Plk1 using plogosertib represents a promising therapeutic approach for advanced CRC.

Sod1 deficiency in mouse oocytes during invitro maturation increases chromosome segregation errors with a reduced BUBR1 at kinetochore.

This study aimed to investigate the molecular mechanisms associated with chromosome segregation errors caused by intrinsic oxidative stress during invitro oocyte maturation (IVM) using oocytes from Sod1-deficient (Sod1KO) mice. Ovulated or invitro matured cumulus-cells oocyte complexes (COCs) were collected from wild-type (WT) and Sod1KO mice and evaluated chromosome alignment, chromosome segregation, meiotic progression, and BUBR1 and REC8 protein expression levels. In 21% O2 IVM, the Sod1KO had significantly higher frequencies of chromosome misalignment and segregation errors compared to the WT, and they also reached Germinal Vesicle Break Down (GVBD) and M I stages peak earlier and showed a shorter M I stage residence time compared to the WT. These changes were associated with a decrease in the recruitment of BUBR1 to kinetochores at M I stage, but there were no differences in the expression of REC8 between the two genotypes. Furthermore, the addition of L-ascorbic acid (AsA) or N-acetyl-L-cysteine (NAC) during IVM reduced the frequency of chromosome segregation errors in Sod1KO oocytes. Oxidative stress caused by Sod1 deficiency during IVM impairs the spindle assembly checkpoint function due to a decrease in the recruitment of BUBR1 to M I stage kinetochores, leading to abnormalities in meiotic progression and chromosome segregation.

The microtubule-severing enzyme spastin regulates spindle dynamics to promote chromosome segregation in Trypanosoma brucei.

Microtubule-severing enzymes play essential roles in regulating diverse cellular processes, including mitosis and cytokinesis, by modulating microtubule dynamics. In the early branching protozoan parasite Trypanosoma brucei , microtubule-severing enzymes are involved in cytokinesis and flagellum length control during different life cycle stages, but none of them have been found to regulate mitosis in any life cycle form. Here, we report the biochemical and functional characterization of the microtubule-severing enzyme spastin in the procyclic form of T. brucei . We demonstrate that spastin catalyzes microtubule severing in vitro and ectopic overexpression of spastin disrupts spindle microtubules in vivo in trypanosome cells, leading to defective chromosome segregation. Knockdown of spastin impairs spindle integrity and disrupts chromosome alignment in metaphase and chromosome segregation in anaphase. We further show that the function of spastin requires the catalytic AAA-ATPase domain, the microtubule-binding domain, and the microtubule interacting and trafficking domain, and that the association of spastin with spindle depends on the microtubule-binding domain. Together, these results uncover an essential role for spastin in chromosome segregation by regulating spindle dynamics in this unicellular eukaryote.

PARL regulates porcine oocyte meiotic maturation by mediating mitochondrial activity.

PARL is a rhomboid membrane protein that plays a crucial role in regulating the metabolism and maintaining the homeostasis of mitochondria which provide important energy and material reserves for oocyte maturation. However, the impact of PARL on oocyte maturation remains poorly understood. Here, we elucidated the pivotal role of PARL in oocyte maturation through its regulatory effects on mitochondrial activity. Specifically, our findings revealed that inhibiting PARL expression by interfering with RNA transcription in oocytes led to a substantial decrease in the rate of first polar body extrusion and early development of parthenogenetically activated embryos. Moreover, PARL deficiency disrupted mitochondrial distribution and activity, leading to the accumulation of ROS, abnormal distribution of CGs and actin, increased tubulin acetylation modification, disturbed spindle assembly and chromosome alignment, ultimately caused DNA damage in porcine oocytes at the metaphase II stage. Intriguingly, PARL deficiency did not cause occurrence of apoptosis in oocytes. Furthermore, our study highlighted that PARL deficiency caused the aberrant expression of genes associated with oocyte maturation, particularly those genes associated with mitochondrial function and DNA integrity. Collectively, these results demonstrate that the indispensable role of PARL in orchestrating porcine oocyte meiotic maturation though its modulation of mitochondrial activity.

A Microtubule-Associated Protein Functions in Preventing Oocytes from Evading the Spindle Assembly Checkpoint.

Aneuploidy eggs are a common cause of human infertility, spontaneous abortion, or trisomy syndromes. The spindle assembly checkpoint (SAC) plays a crucial role in preventing aneuploidy in oocytes, yet it is unclear if additional mechanisms exist to ensure oocyte adherence to this checkpoint. It is now revealed that the microtubule-associated protein NUSAP can prevent oocytes from evading the SAC and regulate the speed of the cell cycle. Mechanistically, the study identifies NUSAP as a novel stabilizer of the E3 ubiquitin ligase APC/CCDH1, protecting CDH1 from SCFBTRC-mediated degradation. Depletion of NUSAP reduces CDH1 protein level, leading to abnormal spindle assembly and chromosome alignment, and disrupting the balance of cell cycle proteins. This misregulated balance causes oocytes to evade the SAC. Consequently, these abnormal oocytes not only fail to arrest at metaphase but also accelerate the cell process, ultimately resulting in the production of aneuploid eggs. Together, the findings not only clarify the existence of mechanisms that ensure oocytes compliance with the spindle assembly checkpoint but also expand the new functions of NUSAP beyond its role as a microtubule- associated protein.

An evolutionary perspective on the relationship between kinetochore size and CENP-E dependence for chromosome alignment.

Chromosome alignment during mitosis can occur as a consequence of bi-orientation or is assisted by the CENP-E (kinesin-7) motor at kinetochores. We previously found that Indian muntjac chromosomes with larger kinetochores bi-orient more efficiently and are biased to align in a CENP-E-independent manner, suggesting that CENP-E dependence for chromosome alignment negatively correlates with kinetochore size. Here, we used targeted phylogenetic profiling of CENP-E in monocentric (localized centromeres) and holocentric (centromeres spanning the entire chromosome length) clades to test this hypothesis at an evolutionary scale. We found that, despite being present in common ancestors, CENP-E was lost more frequently in taxa with holocentric chromosomes, such as Hemiptera and Nematoda. Functional experiments in two nematodes with holocentric chromosomes in which a CENP-E ortholog is absent (Caenorhabditis elegans) or present (Pristionchus pacificus) revealed that targeted expression of human CENP-E to C. elegans kinetochores partially rescued chromosome alignment defects associated with attenuated polar-ejection forces, whereas CENP-E inactivation in P. pacificus had no detrimental effects on mitosis and viability. These data showcase the dispensability of CENP-E for mitotic chromosome alignment in species with larger kinetochores.

Age-associated accumulation of RAB9 disrupts oocyte meiosis.

The critical role of some RAB family members in oocyte meiosis has been extensively studied, but their role in oocyte aging remains poorly understood. Here, we report that the vesicle trafficking regulator, RAB9 GTPase, is essential for oocyte meiosis and aging in humans and mice. RAB9 was mainly located at the meiotic spindle periphery and cortex during oocyte meiosis. In humans and mice, we found that the RAB9 protein level were significantly increased in old oocytes. Age-related accumulation of RAB9 inhibits first polar body extrusion and reduces the developmental potential of oocytes. Further studies showed that increased Rab9 disrupts spindle formation and chromosome alignment. In addition, Rab9 overexpression disrupts the actin cap formation and reduces the cortical actin levels. Mechanically, Rab9-OE increases ROS levels, decreases mitochondrial membrane potential, ATP content and the mtDNA/nDNA ratio. Further studies showed that Rab9-OE activates the PINK1-PARKIN mitophagy pathway. Importantly, we found that reducing RAB9 protein expression in old oocytes could partially improve the rate of old oocyte maturation, ameliorate the accumulation of age-related ROS levels and spindle abnormalities, and partially rescue ATP levels, mtDNA/nDNA ratio, and PINK1 and PARKIN expression. In conclusion, our results suggest that RAB9 is required to maintain the balance between mitochondrial function and meiosis, and that reducing RAB9 expression is a potential strategy to ameliorate age-related deterioration of oocyte quality.

Anapc5 and Anapc7 as genetic modifiers of KIF18A function in fertility and mitotic progression.

The kinesin family member 18A (KIF18A) is an essential regulator of microtubule dynamics and chromosome alignment during mitosis. Functional dependency on KIF18A varies by cell type and genetic context but the heritable factors that influence this dependency remain unknown. To address this, we took advantage of the variable penetrance observed in different mouse strain backgrounds to screen for loci that modulate germ cell depletion in the absence of KIF18A. We found a significant association at a Chr5 locus where anaphase promoting complex subunits 5 (Anapc5) and 7 (Anapc7) were the top candidate genes. We found that both genes were differentially expressed in a sensitive strain background when compared to resistant strain background at key timepoints in gonadal development. We also identified a novel retroviral insertion in Anapc7 that may in part explain the observed expression differences. In cell line models, we found that depletion of KIF18A induced mitotic arrest, which was partially rescued by co-depletion of ANAPC7 (APC7) and exacerbated by co-depletion of ANAPC5 (APC5). These findings suggest that differential expression and activity of Anapc5 and Anapc7 may influence sensitivity to KIF18A depletion in germ cells and CIN cells, with potential implications for optimizing antineoplastic therapies.

Phosphorylation of Ephexin4 at Ser-41 contributes to chromosome alignment via RhoG activation in cell division.

Ephexin proteins are guanine nucleotide exchange factors for the Rho GTPases. We reported that Ephexin4 regulates M-phase progression downstream of phosphorylated EphA2, a receptor-type tyrosine kinase, through RhoG activation; however, the regulation of Ephexin4 during M phase remains unknown. In this study, a novel Ephexin4 phosphorylation site was identified at Ser41, exclusively in M phase. Ephexin4 knockdown prolonged the duration of M phase by activating the spindle assembly checkpoint, at which BubR1 was localized at the kinetochores of the misaligned chromosomes. This delay was alleviated by re-expression of wild-type, but not S41A Ephexin4. The Ephexin4 knockdown caused chromosome misalignment and reduced the RhoG localization to the plasma membrane. These phenotypes were rescued by re-expression of wild-type and phospho-mimic S41E mutant, but not the S41A mutant. Consistently, S41E mutant enhanced active RhoG levels, even in the interphase. Regardless of the Ephexin4 knockdown, active RhoG-G12V was localized at the plasma membrane. Furthermore, Ephexin4 knockdown exacerbated vincristine-induced chromosome misalignment, which was prevented by re-expressing the wild-type but not S41A Ephexin4. Overexpression of wild-type and S41E mutant, but not S41A mutant, resulted in an increased number of Madin-Darby canine kidney (MDCK) cysts with cells inside the lumen, indicating disruption of epithelial morphogenesis by deregulating Ephexin4/RhoG signaling in cell division. Our results suggest that Ephexin4 undergoes phosphorylation at Ser41 in cell division, and the phosphorylation is required for chromosome alignment through RhoG activation. Combined with mitosis-targeting agents, inhibition of Ephexin4 phosphorylation may represent a novel strategy for cancer chemotherapy.

4D Microscopy and Tracking of Chromosomes and the Spindle in C. elegans Early Embryos.

Maintaining genomic integrity throughout successive cell divisions is essential for the proper development and functioning of organisms. Chromosome alignment and segregation occur on a microtubule-based spindle originating from centrosomes. The molecular and cellular mechanisms involved in accurate chromosome segregation during early embryonic divisions are highly conserved between worms and humans. Therefore, C. elegans serves as a robust model for investigating mitotic cell divisions within a metazoan system. Throughout early embryonic development, filming and tracking successive cell divisions becomesprogressively more challenging as the number of cells increases and cell size decreases. To address this challenge, we describe a method for preparing live samples, performing 4D time-lapse imaging, and semi-automated tracking of chromosomes and spindle poles during early mitotic divisions in C. elegans embryos.

Anti-Centromere Protein A Antibody Disrupts the Competence of Mouse Oocytes Matured In Vitro.

Anticentromere autoantibodies are associated with refractory IVF/ET failure, but causality is unclear. Experimental models are needed. Immature oocytes collected from 23-day-old mice were matured in vitro for 18h in a culture medium containing an anti-human centromere protein A (CENP-A) polyclonal antibody, and those oocytes' maturity and chromosome/spindle structure were assessed. Antibody exposure did not affect the germinal vesicle breakdown ratio but reduced the first polar body formation ratio by 13% at the highest concentration (70.0 µg/mL). Metaphase II (MII) oocytes were stained for chromosomes/spindles and grouped into aligned/barrel-like (AB), scattered/weakly-stained (SW), and condensed/absent (CA). Antibody exposure decreased AB and increased SW and CA in a dose-dependent manner. The AB/SW/CA percentages were 86/14/0, 86/14/0, 35/65/0, and 0/0/100 in the 0, 17.5, 35.0, and 70.0 µg/mL antibody groups, respectively (underlined values represent p < 0.05 compared with 0 µg/mL). Next, metaphase II oocytes were subjected to intracytoplasmic sperm injection, and the number of pronucleus/pronuclei (PN) was counted 6 h later. Antibody exposure decreased two pronuclei oocytes and increased non-two pronuclei oocytes dose-dependently. The percentages of 0/1/2/3 pronuclei oocytes were 43/0/57/0, 37/0/21/42, 16/28/48/8, and 91/4/4/0 in the 0, 17.5, 35.0, and 70.0µg/mL groups, respectively. Anti-CENP-A antibody impaired a linear alignment of chromosomes at metaphase II and enhanced one or three PN formation after ICSI, which are similar to findings reported for infertile women with anticentromere autoantibodies.

Identification of antimitotic sulfonamides inhibiting chromosome congression.

The discovery of new small-molecule inhibitors is essential to enhancing our understanding of biological events at the molecular level and driving advancements in drug discovery. Mitotic inhibitors have played a crucial role in development of anticancer drugs. Beyond traditional microtubule inhibitors, various inhibitors targeting specific mitotic factors have been developed. This study aimed to develop novel mitotic inhibitors targeting chromosome alignment. We established a cell-based screening method using Cell Division Cycle Associated 5 (CDCA5) and kinesin-5 as markers, designed to efficiently detect mitotic phenotypes characterized by aberrant bipolar spindles with some misaligned chromosomes. Through this screening, we identified CAIS-1, an aryl sulfonamide with unique antimitotic properties. CAIS-1 exhibits dual functionality by inhibiting chromosome congression at low concentrations and spindle microtubule formation at high concentrations, causing a concentration-dependent mitotic arrest, followed by apoptotic cell death. Mechanistic studies revealed that CAIS-1 directly acts on tubulin at high concentrations, thereby inhibiting tubulin polymerization in vitro. In contrast, at low concentrations, CAIS-1 functions through a mechanism distinct from GSK923295, a conventional chromosome congression inhibitor targeting Centromere-associated protein-E (CENP-E), highlighting its unique mode of action. Moreover, CAIS-2, a structural analog of CAIS-1, selectively inhibits chromosome congression without significantly affecting spindle microtubules. This observation suggests that CAIS-1 and CAIS-2 function as antimitotic sulfonamides with distinct targets beyond tubulin, thus offering additional biological potential of sulfonamide compounds. Together, CAIS-1 and CAIS-2 represent promising tools for providing new molecular insights into kinetochore function during mitosis and for exploring new approaches in anticancer drug development.

Acentric chromosome congression and alignment on the metaphase plate via kinetochore-independent forces in Drosophila.

Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in Drosophila neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently congress and align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in the presence of spindles with disrupted interpolar microtubules acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer interpolar microtubules contribute significantly to acentric congression and alignment. Surprisingly, we observe disrupted telomere pairing and alignment of sister acentrics indicating that the kinetochore is required to ensure proper gene-to-gene alignment of sister chromatids. Finally, we demonstrate that like mammalian cells, the Drosophila congressed chromosomes on occasion exhibit a toroid configuration.

Microtubule poleward flux as a target for modifying chromosome segregation errors

Cancer cells often display errors in chromosome segregation, some of which result from improper chromosome alignment at the spindle midplane. Chromosome alignment is facilitated by different rates of microtubule poleward flux between sister kinetochore fibers. However, the role of the poleward flux in supporting mitotic fidelity remains unknown. Here, we introduce the hypothesis that the finely tuned poleward flux safeguards against lagging chromosomes and micronuclei at mitotic exit by promoting chromosome alignment in metaphase. We used human untransformed RPE-1 cells depleted of KIF18A/kinesin-8 as a system with reduced mitotic fidelity, which we rescued by three mechanistically independent treatments, comprising low-dose taxol or codepletion of the spindle proteins HAUS8 or NuMA. The rescue of mitotic errors was due to shortening of the excessively long overlaps of antiparallel microtubules, serving as a platform for motor proteins that drive the flux, which in turn slowed down the overly fast flux and improved chromosome alignment. In contrast to the prevailing view, the rescue was not accompanied by reduction of overall microtubule growth rates. Instead, speckle microscopy revealed that the improved chromosome alignment in the rescue treatments was associated with slower growth and flux of kinetochore microtubules. In a similar manner, a low-dose taxol treatment rescued mitotic errors in a high-grade serous ovarian carcinoma cell line OVKATE. Collectively, our results highlight the potential of targeting microtubule poleward flux to modify chromosome instability and provide insight into the mechanism through which low doses of taxol rescue certain mitotic errors in cancer cells.

4D live tracing reveals distinct movement trajectories of meiotic chromosomes.

Proper chromosome alignment at the spindle equator is a prerequisite for accurate chromosome segregation during cell division. However, the chromosome movement trajectories prior to alignment remain elusive. Here, we established a 4D imaging analysis framework to visualize chromosome dynamics and develop a deep-learning model for chromosome movement trajectory classification. Our data reveal that chromosomes follow at least three distinct movement trajectories (retracing, congressing, and quasi-static) to arrive at the equator. We further revealed the distinct roles of multiple kinesin superfamily proteins (KIFs) in coordinating and maintaining the chromosome movement trajectories. In summary, we have presented an efficient and unbiased approach to studying chromosome dynamics during cell division, thereby uncovering a variety of chromosome movement trajectories that precede alignment.

HURP regulates Kif18A recruitment and activity to synergistically control microtubule dynamics

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determine the binding mode of HURP to microtubules using cryo-EM. The structure helps rationalize why HURP functions as a microtubule stabilizer. Additionally, HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observe that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in microtubule length control.

Mutation of the SUMOylation site of Aurora-B disrupts spindle formation and chromosome alignment in oocytes

Aurora-B is a kinase that regulates spindle assembly and kinetochore-microtubule (KT-MT) attachment during mitosis and meiosis. SUMOylation is involved in the oocyte meiosis regulation through promoting spindle assembly and chromosome segregation, but its substrates to support this function is still unknown. It is reported that Aurora-B is SUMOylated in somatic cells, and SUMOylated Aurora-B contributes the process of mitosis. However, whether Aurora-B is SUMOylated in oocytes and how SUMOylation of Aurora-B impacts its function in oocyte meiosis remain poorly understood. In this study, we report that Aurora-B is modified by SUMOylation in mouse oocytes. The results show that Aurora-B colocalized and interacted with SUMO-2/3 in mouse oocytes, confirming that Aurora-B is modified by SUMO-2/3 in this system. Compared with that in young mice, the protein expression of SUMO-2/3 decreased in the oocytes of aged mice, indicating that SUMOylation might be related to mouse aging. Overexpression of Aurora-B SUMOylation site mutants, Aurora-BK207R and Aurora-BK292R, inhibited Aurora-B recruitment and first polar body extrusion, disrupting localization of gamma tubulin, spindle formation and chromosome alignment in oocytes. The results show that it was related to decreased recruitment of p-HDAC6 which induces the high stability of whole spindle microtubules including the microtubules of both correct and wrong KT-MT attachments though increased acetylation of microtubules. Therefore, our results corroborate the notion that Aurora-B activity is regulated by SUMO-2/3 in oocytes, and that SUMOylated Aurora B plays an important role in spindle formation and chromosome alignment.

Reduced NET1 adversely affects early embryonic development in mice

Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily-specific guanine/nucleotide-exchange factor that exhibits critical roles in diverse biological processes. However, the functions in mouse preimplantation embryonic development have not yet determined. In the present study we demonstrated that NET1 is a key factor in the outcome of early mouse embryonic development. Immunofluorescence detection showed that NET1 is principally localized to the nucleus during mouse pre-implantation embryonic development. Silencing Net1 at the zygote stage using a specific siRNA impaired the developmental competence of early mouse embryos, and Net1-knockdown (Net1-KD) induced mitotic spindle-assembly defects and chromosomal alignment abnormalities at the first embryonic cleavage. In addition, reduced NET1 exacerbated reactive oxygen species production and DNA lesions in two-cell stage embryos, further augmenting cellular apoptosis in the preimplantation blastocyst. In summary, our data display key roles for NET1 in mitotic spindle assembly, oxidative stress, and DNA damage during early mouse embryonic development.

Alignment of a Trivalent Chromosome on the Metaphase Plate Is Associated with Differences in Microtubule Density at Each Kinetochore.

Chromosome alignment on the metaphase plate is a conserved phenomenon and is an essential function for correct chromosome segregation for many organisms. Organisms with naturally-occurring trivalent chromosomes provide a useful system for understanding how chromosome alignment is evolutionarily regulated, as they align on the spindle with one kinetochore facing one pole and two facing the opposite pole. We studied chromosome alignment in a praying mantid that has not been previously studied chromosomally, the giant shield mantis Rhombodera megaera. R. megaera has a chromosome number of 2n = 27 in males. Males have X1, X2, and Y chromosomes that combine to form a trivalent in meiosis I. Using live-cell imaging of spermatocytes in meiosis I, we document that sex trivalent Y chromosomes associate with one spindle pole and the two X chromosomes associate with the opposing spindle pole. Sex trivalents congress alongside autosomes, align with them on the metaphase I plate, and then the component chromosomes segregate alongside autosomes in anaphase I. Immunofluorescence imaging and quantification of brightness of kinetochore-microtubule bundles suggest that the X1 and X2 kinetochores are associated with fewer microtubules than the Y kinetochore, likely explaining the alignment of the sex trivalent at the spindle equator with autosomes. These observations in R. megaera support the evolutionary significance of the metaphase alignment of chromosomes and provide part of the explanation for how this alignment is achieved.

Bisphenol AP inhibits mouse oocyte maturation in vitro by disrupting cytoskeleton architecture and cell cycle processes

Bisphenol A (BPA) is among the extensively researched environmental endocrine-disrupting chemicals (EDCs), and its utilization is restricted owing to the detrimental impacts it has on human health. Bisphenol AP (BPAP) is one of the alternatives to BPA, but the influence of BPAP on human health has not been elucidated. The objective of the current research was to determine the influence of BPAP exposure on the in vitro maturation of mouse oocytes and to explore its potential reproductive toxicity. BPAP exposure was found to inhibit polar body extrusion during mouse oocyte maturation, resulting in an arrest at the metaphase I stage of meiosis. Exposure to BPAP led to sustained activation of BubR1, preventing the degradation of both Securin and Cyclin B1. Mechanistically, BPAP exposure disrupts spindle assembly and chromosome alignment. Levels of acetylated α-tubulin were significantly elevated in BPAP-treated oocytes, reflecting decreased spindle stability. Exposure to BPAP also induced DNA damage and impaired DNA damage repair. In addition, BPAP exposure altered histone modification levels. In summary, this investigation suggests that exposure to BPAP can influence cytoskeletal assembly, interfere with cell cycle progression, induce DNA damage, alter histone modifications, and ultimately impede oocyte meiotic maturation. This investigation enhances understanding of the impact of bisphenol analogs on female gametes, underscoring that BPAP cannot be considered a reliable replacement for BPA.