Chromosome Segregation Errors Research Articles

Targeting chromosomal instability in patients with cancer.

Chromosomal instability (CIN) is a hallmark of cancer and a driver of metastatic dissemination, therapeutic resistance, and immune evasion. CIN is present in 60-80% of human cancers and poses a formidable therapeutic challenge as evidenced by the lack of clinically approved drugs that directly target CIN. This limitation in part reflects a lack of well-defined druggable targets as well as a dearth of tractable biomarkers enabling direct assessment and quantification of CIN in patients with cancer. Over the past decade, however, our understanding of the cellular mechanisms and consequences of CIN has greatly expanded, revealing novel therapeutic strategies for the treatment of chromosomally unstable tumours as well as new methods of assessing the dynamic nature of chromosome segregation errors that define CIN. In this Review, we describe advances that have shaped our understanding of CIN from a translational perspective, highlighting both challenges and opportunities in the development of therapeutic interventions for patients with chromosomally unstable cancers.

O-008 Sperm-derived centrioles play a key role in ensuring correct chromosome segregation in the first mitotic division of mammalian zygotes

Abstract Study question What is the role of the centrosomes during mitotic division in the mammalian zygote? Does the atypical structure of sperm-derived centrioles affect their function? Summary answer Correct centrosome positioning guarantees spindle stability and correct chromosome segregation. Centrosome malposition or inactivity predisposes to chromosome segregation errors and failure of cytokinesis. What is known already The centrosomes of somatic cells comprise two centrioles surrounded by pericentriolar material, and are major microtubule-organizing centres with an essential role in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and, although two centrioles are introduced by the spermatozoon at fertilization, they are structurally different to the canonical centrioles found in somatic cells. We recently demonstrated that centrosomes play a minor role during zygotic spindle assembly, when they are often mal-positioned (not at the spindle poles). However, the causes and consequences of abnormal centrosome positioning in zygotes are not known. Study design, size, duration We combined systematic immunofluorescence of bovine zygotes, fixed at different stages, with real-time imaging of live zygotes to investigate the implications of centrosome mal-positioning during zygotic mitosis on the fidelity of chromosome segregation and cytokinesis. To study the functionality of the sperm-derived centrioles alone, we inhibited the formation of daughter centrioles using the PLK4 inhibitor (PLK4i), Centrinone. Bovine zygotes were used because the structure of bovine and human sperm-derived centrioles are very similar. Participants/materials, setting, methods Bovine oocytes were retrieved from slaughterhouse ovaries and matured in vitro. Matured oocytes were fertilized in vitro with semen from a bull of proven fertility and, either fixed at 27-29h post-fertilization, or used for time-lapse light-sheet imaging after injection of mRNA for microtubule and chromatin reporters. Fixed bovine zygotes were immunostained for chromatin, alpha-tubulin, centriolar and pericentriolar components, and imaged with a confocal microscope. Microtubule nucleation ability was evaluated using a microtubule regrowth assay. Main results and the role of chance Live imaging showed that in 52% of bovine zygotes (24/46) the centrosomes engaged with the spindle but were malposition, while in 26% (12/46) the centrosomes were not visible (inactive). All zygotes with normally positioned centrosomes showed correct chromosome segregation. Centrosome malposition and inactivity induced chromosome segregation errors in 44% and 82% of the zygotes respectively. PLK4i treatment successfully inhibited centriole duplication, since the volume of core-centriole protein (POC5) in treated zygotes was half that in non-treated zygotes. When centriole duplication was prevented, the sperm-derived centrioles showed a similar ability to recruit pericentrosomal material (NEDD1) and nucleate microtubules. However, live imaging showed that 82% (14/17) of PLK4i treated zygotes the spindle, although bipolar, was unusually elongated and chromosomes either failed to align correctly on the metaphase plate or showed anaphase lag. Moreover, in treated zygotes where both centrioles were positioned at the same spindle pole, the spindle failed to anchor correctly to the cell membrane and resulted in rotational behavior of the zygote and an increase of cytokinesis failure from up to 47% (8/17). Therefore we conclude that centrosomes are likely to play a key role in correct chromosome segregation in mammalian zygotes. Limitations, reasons for caution For ethical reasons, we could not manipulate and study human zygotes. Instead, we chose an animal model with a rate of embryonic aneuploidy similar to man. It is, however, possible that zygotic centrosome function differs between these two species. Wider implications of the findings A better understanding of the key regulators of centrosome function might lead to the development of interventions to reduce the risk of malfunction, thereby reducing the incidence of embryonic aneuploidy. Moreover, our study unveiled an mechanism by which impaired PLK4 function induces aneuploidy in mammalian embryos. Trial registration number not applicable

P-558 Characterization of the morphology of the outer-kinetochore expandable module, aka fibrous corona, and its role in ensuring proper chromosome segregation in human and bovine oocytes

Abstract Study question How does the Fibrous Corona (FC) behave in human and bovine oocyte meiosis and what is its function in chromosome segregation during meiosis I? Summary answer In human and bovine oocytes, FC shows extreme plasticity expanding beyond the centromeric region of the chromosomes. Pharmacological inhibition of FC’s expansion induces chromosome mis-segregation. What is known already Embryonic aneuploidy of meiotic origin is one of the major causes of implantation failure and early pregnancy loss. Erroneous microtubule (MT)-kinetochore attachments in human meiosis are the main cause of meiotic chromosome segregation errors. In somatic cells, the FC, a multiprotein complex, assembles on kinetochores and expands around the centromere during prometaphase to facilitate MT interactions and stimulate correct MT-kinetochore attachment. We have shown that in bovine oocytes FC expand beyond the centrosomes and is a potent MTs nucleator (10.1016/j.cub.2023.01.010). However, it is still unknown how FC behaves and what are its functions in MT-kinetochore interactions in human oocytes. Study design, size, duration FC morphology was studied in immature human oocytes discarded during ICSI procedures and donated (informed consent) by the patients for research (Ethical permit TCBio.21-754) during the years 2021-2024. Because of ethical concerns and reduced availability of human oocytes, functional studies on FC were performed on bovine oocytes collected from slaughterhouse ovaries. Participants/materials, setting, methods 180 immature human oocytes were matured in vitro and either fixed at different meiosis I stages or treated 2h with colcemid, a MT depolymerizing drug, to induce maximum FC expansion and fixed. The oocytes were then immuno-stained for chromatin(DAPI), FC(ZW10), MT(alpha tubulin) and kinetochores(CREST) and imaged with super-resolution confocal microscopy. Bovine immature oocytes were matured in vitro with or without lonafarnib (inhibitor of FC expansion). After maturation chromosome spreads were performed to evaluate oocyte ploidy. Main results and the role of chance In human oocytes, after nuclear envelope breakdown, before microtubule appearance when the chromosomes appear compacted, FCs are visible on the top of the kinetochores. During MT nucleation, FCs form thick mesh in the center of the compacted chromosomes. During prometaphase I, FCs expand beyond the centromeric region of the chromosomes forming a cup around the sister kinetochores of the paired homologous chromosomes. When the chromosomes starts to align at the metaphase plate, FCs start to be stripped from the kinetochores and colocalize to the MTs. At metaphase I and II, only kinetochore localization is visible. When oocytes are treated with colcemid, FCs extend to partially cover chromosome arms. We observed a wide variability in FCs expansion between and within patients. Inhibition of FC expansion in bovine oocytes increase the incidence of oocyte aneuploidy (90% in lonafarnib treated vs 37% in control oocytes). Altogether, our data reveal that in human oocytes FCs expand more compared to human somatic cells. Considering that inhibition of FC expansion induces chromosome segregation errors, we propose that the large FC size in human oocytes may play a role in achieving correct chromosome-MT attachments within their extremely large cytoplasm thus ensuring proper chromosome segregation. Limitations, reasons for caution The limited availability of human oocytes requires the use of an animal model for in depth mechanistic studies. Although bovine oocytes have similar aneuploidy rate and spindle assembly mechanisms as human oocytes, they show morphological differences in FC expansion which could underlie intrinsic functional discrepancies between the two organisms. Wider implications of the findings A better understanding of the chromosome segregation mechanisms in human meiosis will help understanding what causes the high incidence of aneuploidies in human oocytes and possibly contribute to the development of interventions or of oocyte selection procedures to reduce the incidence of oocyte aneuploidy during in vitro maturation. Trial registration number Not applicable

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

A conserved site on Ndc80 complex facilitates dynamic recruitment of Mps1 to yeast kinetochores to promote accurate chromosome segregation

Accurate chromosome segregation relies on kinetochores carrying out multiple functions, including establishing and maintaining microtubule attachments, forming precise bi-oriented attachments between sister chromatids, and activating the spindle assembly checkpoint. Central to these processes is the highly conserved Ndc80 complex. This kinetochore subcomplex interacts directly with microtubules but also serves as a critical platform for recruiting kinetochore-associated factors and as a key substrate for error correction kinases. The precise manner in which these kinetochore factors interact and regulate each other's function remains unknown, considerably hindering our understanding of how Ndc80 complex-dependent processes function together to orchestrate accurate chromosome segregation. Here, we aimed to uncover the role of Nuf2's CH domain, a component of the Ndc80 complex, in ensuring these processes. Through extensive mutational analysis, we identified a conserved interaction domain composed of two segments in Nuf2's CH domain that form the binding site for Mps1 within the yeast Ndc80 complex. Interestingly, this site also associates with the Dam1 complex, suggesting Mps1 recruitment may be subject to regulation by competitive binding with other factors. Mutants disrupting this "interaction hub" exhibit defects in spindle assembly checkpoint function and severe chromosome segregation errors. Significantly, specifically restoring Mps1-Ndc80 complex association rescues these defects. Our findings shed light on the intricate regulation of Ndc80 complex-dependent functions and highlight the essential role of Mps1 in kinetochore bi-orientation and accurate chromosome segregation.

Chromothripsis: an emerging crossroad from aberrant mitosis to therapeutic opportunities.

Chromothripsis, a type of complex chromosomal rearrangement originally known as chromoanagenesis, has been a subject of extensive investigation due to its potential role in various diseases, particularly cancer. Chromothripsis involves the rapid acquisition of tens to hundreds of structural rearrangements within a short period, leading to complex alterations in one or a few chromosomes. This phenomenon is triggered by chromosome missegregation during mitosis. Errors in accurate chromosome segregation lead to formation of aberrant structural entities such as micronuclei or chromatin bridges. The association between chromothripsis and cancer has attracted significant interest, with potential implications for tumorigenesis and disease prognosis. This review aims to explore the intricate mechanisms and consequences of chromothripsis, with a specific focus on its association with mitotic perturbations. Herein, we discuss a comprehensive analysis of crucial molecular entities and pathways, exploring the intricate roles of the CIP2A-TOPBP1 complex, micronuclei formation, chromatin bridge processing, DNA damage repair, and mitotic checkpoints. Moreover, the review will highlight recent advancements in identifying potential therapeutic targets and the underlying molecular mechanisms associated with chromothripsis, paving the way for future therapeutic interventions in various diseases.

Micronuclei induced by radiation, replication stress, or chromosome segregation errors do not activate cGAS-STING

The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.

A cytological F1 RNAi screen for defects in Drosophila melanogaster female meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, we have screened the VALIUM22 collection of RNAi constructs that target germline-expressing genes in a vector optimized for germline expression by driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4). This allowed us to test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we could identify defects in sterile females. After screening >1,450 lines of the collection for two different defects (chromosome congression and the hypoxic sequestration of Mps1-GFP to ooplasmic filaments), we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

Abstract 6598: Inhibition of cGAS as a strategy to restore anti-tumor immunity response to STING agonist in chromosomally unstable tumors

Abstract The activation of STING (stimulator of interferon genes) has been widely shown to induce signaling pathways, triggering type I interferon and ultimately generating anti-tumor immunity. Whereas initial preclinical studies on STING agonists exhibited boost in anti-tumor response, clinical trials combining STING agonist and immune checkpoint blockade agent PD-1 reported that only small subset of patients benefited from the treatment. This study highlights a potential key mechanism underlying the lack of benefit from STING agonist observed in most clinical trial patients. We reveal a phenomenon known as chromosomal instability (CIN), characterized by persistent chromosome segregation errors over multiple rounds of mitosis, drives chronic activation of cGAS (cyclic GMP‒AMP synthase) through continuously supplying its substrate in the form of micronuclei. Chronic activation of cGAS stimulates degradation of its downstream target STING in a feedback-loop manner. This consequently attenuates type I interferon signaling upon STING activation, blunting its anti-tumor response. Remarkably, we show suppression of CIN, achieved through introduction of mitotic centromere-associated kinesin (MCAK), or inhibition of cGAS activity, either by pharmacological agent or total genetic ablation of cGAS using CRISPR-Cas9, restores type I interferon production and anti-tumor immunity upon the administration of STING agonist, MSA-2 in four of the five advanced tumor models. Furthermore, we show this restoration of type I interferon signaling and anti-tumor immunity is cancer cell STING-dependent as genetic deletion of STING in the tumor cells nullifies this effect. In summary, we provide a mechanistic insight underlying the limited anti-tumor response observed in STING agonist trials and propose potential strategies, through finetuning of CIN level or inhibition of cGAS activity, to illicit stronger anti-tumor immunity in combination with STING agonist treatment. Citation Format: Christy Hong, Lindsay Caprio, Mercedes Duran, Meri Rogava, Hsiang-His Ling, Sreeharsha Gurrapu, Leon Ebel, Melody Di Bona, Benjamin Izar, Samuel F. Bakhoum. Inhibition of cGAS as a strategy to restore anti-tumor immunity response to STING agonist in chromosomally unstable tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6598.

Early onset of APC/C activity renders SAC inefficient in mouse embryos.

Control mechanisms of spindle assembly and chromosome segregation are vital for preventing aneuploidy during cell division. The mammalian germ cells and embryos are prone to chromosome segregation errors, and the resulting aneuploidy is a major cause of termination of development or severe developmental disorders. Here we focused on early mouse embryos, and using combination of methods involving microinjection, immunodetection and confocal live cell imaging, we concentrated on the Spindle Assembly Checkpoint (SAC) and Anaphase Promoting Complex/Cyclosome (APC/C). These are two important mechanisms cooperating during mitosis to ensure accurate chromosome segregation, and assessed their activity during the first two mitoses after fertilization. Our results showed, that in zygotes and 2-cell embryos, the SAC core protein Mad1 shows very low levels on kinetochores in comparison to oocytes and its interaction with chromosomes is restricted to a short time interval after nuclear membrane disassembly (NEBD). Exposure of 2-cell embryos to low levels of spindle poison does not prevent anaphase, despite the spindle damage induced by the drug. Lastly, the APC/C is activated coincidentally with NEBD before the spindle assembly completion. This early onset of APC/C activity, together with precocious relocalization of Mad1 from chromosomes, prevents proper surveillance of spindle assembly by SAC. The results contribute to the understanding of the origin of aneuploidy in early embryos.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse? The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI). Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy. Cumulus-oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23). We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body-egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1-3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished. We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001). N/A. We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability. We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity. This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.

Securin acetylation prevents precocious separase activation and premature sister chromatid separation

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

The spindle protein CKAP2 regulates microtubule dynamics and ensures faithful chromosome segregation

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation.

Chromosomal, Gestational, and Neonatal Outcomes of Embryos Classified as a Mosaic by Preimplantation Genetic Testing for Aneuploidy

(Abstracted from Fertil Steril 2023;120(5):957) Chromosomal segregation errors during mitosis can result in embryonic mosaicism, a phenomenon where different chromosomal copy numbers exist within a single embryo. Preimplantation genetic testing for aneuploidy (PGT-A) of multicellular biopsies can produce intermediate copy numbers in these cases, where some cells are disomic and some show monosomy or trisomy.

Intracellular enrichment of lipid droplets increases cytoskeletal polymerization and chromosome segregation errors

Intracellular enrichment of lipid droplets increases cytoskeletal polymerization and chromosome segregation errors

Abstract IA020: Micronuclei are a nexus of DNA damage signaling and genomic instability

Abstract Micronuclei have classically been considered inert biomarkers of genomic instability arising from failed DNA damage repair and chromosome segregation errors in mitosis. Work from several groups including our own have now demonstrated that micronuclei are active contributors to the cellular response to DNA damage by driving chromothripsis and through induction of anti-viral gene expression programs mediated by the cGAS-STING pathway. We now show that the specific DNA damage type leading to micronuclei formation influences the capacity for cGAS localization and activation. This arises in part due to discrete chromatin features, present prior to DNA damage induction, that are retained in micronuclei several days after their formation. Together these data demonstrate that micronuclei connect the pre-damage chromatin status and the chromatin context of micronuclei-inciting lesions to the long-term signaling outcomes important for anti-tumor immunity. This finding implies that micronuclei represent archives for the nature of DNA repair failure and downstream signaling consequences. Based on this premise we will present our efforts in micronuclear proteomics where we uncover common features of micronuclei including splicing deficiencies. Such deficiencies presage exacerbated MN-associated DNA damage that is upstream of chromothripsis. These studies have direct implications for how cells sense the aftermath of DNA damage and how micronuclei contribute to ongoing genomic instability. Citation Format: Shane M. Harding. Micronuclei are a nexus of DNA damage signaling and genomic instability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA020.

MAARS Software for Automatic and Quantitative Analysis of Mitotic Progression.

In this chapter, we describe a software called MAARS (Mitotic Analysis And Recording System) that enables automatic and quantitative analysis of mitotic progression on an open-source platform. This computer-assisted analysis of cell division allows the unbiased acquisition of multiple parameters such as cell shape or size, metaphase or anaphase delays, as well as various mitotic abnormalities. This chapter describes the power of such an expert system to highlight the complexity of the mechanisms required to prevent mitotic chromosome segregation errors, leading to aneuploidy.

Effect of ovarian stimulation on developmental speed of preimplantation embryo in a mouse model.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

Characterizing chromosomal instability-driven cancer evolution and cell fitness at a glance.

Chromosomal instability (CIN), an increased rate of chromosome segregation errors during mitosis, is a hallmark of cancer cells. CIN leads to karyotype differences between cells and thus large-scale heterogeneity among individual cancer cells; therefore, it plays an important role in cancer evolution. Studying CIN and its consequences is technically challenging, but various technologies have been developed to track karyotype dynamics during tumorigenesis, trace clonal lineages and link genomic changes to cancer phenotypes at single-cell resolution. These methods provide valuable insight not only into the role of CIN in cancer progression, but also into cancer cell fitness. In this Cell Science at a Glance article and the accompanying poster, we discuss the relationship between CIN, cancer cell fitness and evolution, and highlight techniques that can be used to study the relationship between these factors. To that end, we explore methods of assessing cancer cell fitness, particularly for chromosomally unstable cancer.