CENP-C-targeted PLK-1 regulates kinetochore function in C. elegans embryos.

Polo-like kinase 1 (PLK-1) is present in centrosomes, the nuclear envelope, and kinetochores and plays a significant role in meiosis and mitosis. PLK-1 depletion or inhibition has severe consequences for spindle assembly, spindle assembly checkpoint (SAC) activation, chromosome segregation, and cytokinesis. BUB-1 targets PLK-1 to the outer kinetochore and, in mammals, the inner kinetochore PLK1 targeting is mediated by the constitutive centromere associated network (CCAN). BUB1-targeted PLK-1 plays a key role in SAC activation and a SAC-independent role through targeting CDC-20. In contrast, whether there is a specific, non-redundant role for inner kinetochore targeted PLK-1 is unknown. Here, we used the C. elegans embryo to study the role of inner kinetochore PLK-1. We found that CENP-C, the sole CCAN component in C. elegans and other species, targets PLK-1 to the inner kinetochore during prometaphase and metaphase. Disruption of the CENP-C/PLK-1 interaction leads to an imbalance in kinetochore components and a defect in chromosome congression, without affecting CDC-20 recruitment. These findings indicate that PLK-1 kinetochore recruitment by CENP-C has at least partially distinct functions than outer kinetochore PLK-1, providing a platform for a better understanding of the different roles played by PLK-1 during mitosis.

Aligning genotyping and copy number data in single trophectoderm biopsies for aneuploidy prediction: uncovering incomplete concordance.

STUDY QUESTION: To what extent can genotype analysis aid in the classification of (mosaic) aneuploid embryos diagnosed through copy number analysis of a trophectoderm (TE) biopsy? SUMMARY ANSWER: In a small portion of embryos, genotype analysis revealed signatures of meiotic or uniform aneuploidy in those diagnosed with intermediate copy number changes, and signatures of presumed mitotic or putative mosaic aneuploidy in those diagnosed with full copy number changes. WHAT IS KNOWN ALREADY: Comprehensive chromosome screening (CCS) for preimplantation genetic testing has provided valuable insights into the prevalence of (mosaic) chromosomal aneuploidy at the blastocyst stage. However, diagnosis of (mosaic) aneuploidy often relies solely on (intermediate) copy number analysis of a single TE biopsy. Integrating genotype information allows for independent assessment of the origin and degree of aneuploidy. Yet, studies aligning both datasets to predict (putative mosaic) aneuploidy in embryos remain scarce. STUDY DESIGN SIZE DURATION: A single TE biopsy was collected from 1560 embryos derived from 221 couples tested for a monogenic disorder (n = 218) or microdeletion-/microduplication syndrome (n = 3). TE samples were subjected to both copy number and genotyping analysis. PARTICIPANTS/MATERIALS SETTING METHODS: Copy number and SNP genotyping analysis were conducted using GENType. Unbalanced chromosomal anomalies ≥10 Mb (or ≥20 Mb for copy number calls <50%) were classified by degree, based on low-range intermediate (LR, 30-50%), high-range intermediate (HR, 50-70%) or full (>70%) copy number changes. These categories were further subjected to genotyping analysis to ascertain the origin (and/or degree) of aneuploidy. For chromosomal gains, the meiotic division of origin (meiotic I/II versus non-meiotic or presumed mitotic) was established by studying the haplotypes. The level of monosomy (uniform versus putative mosaic) in the biopsy could be ascertained from the B-allele frequencies. For segmental aneuploidies, genotyping was restricted to deletions. MAIN RESULTS AND THE ROLE OF CHANCE: Of 1479 analysed embryos, 24% (n = 356) exhibited a whole-chromosome aneuploidy, with 19% (n = 280) showing full copy number changes suggestive of uniform aneuploidy. Among 258 embryos further investigated by genotyping, 95% of trisomies with full copy number changes were identified to be of meiotic origin. For monosomies, a complete loss of heterozygosity (LOH) in the biopsy was observed in 97% of cases, yielding a 96% concordance rate at the embryo level (n = 248/258). Interestingly, 4% of embryos (n = 10/258) showed SNP signatures of non-meiotic gain or putative mosaic loss instead. Meanwhile, 5% of embryos (n = 76/1479) solely displayed HR (2.5%; n = 37) or LR (2.6%; n = 39) intermediate copy number changes, with an additional 2% showing both intermediate and full copy number changes. Among embryos with HR intermediate copy number changes where genotyping was feasible (n = 25/37), 92% (n = 23/25) showed SNP signatures consistent with putative mosaic aneuploidy. However, 8% (n = 2/25) exhibited evidence of meiotic trisomy (9%) or complete LOH in the biopsy (7%). In the LR intermediate group, 1 of 33 (3%) genotyped embryos displayed complete LOH. Furthermore, segmental aneuploidy was detected in 7% of embryos (n = 108/1479) (or 9% (n = 139) with added whole-chromosome aneuploidy). These errors were often (52%) characterized by intermediate copy number values, which closely aligned with genotyping data when examined (94-100%). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The findings were based on single TE biopsies and the true extent of mosaicism was not validated through embryo dissection. Moreover, evidence of absence of a meiotic origin for a trisomy should not be construed as definitive proof of a mitotic error. Additionally, a genotyping diagnosis was not always attainable due to the absence of a recombination event necessary to discern between meiotic II and non-meiotic trisomy, or the unavailability of DNA from both parents. WIDER IMPLICATIONS OF THE FINDINGS: Interpreting (intermediate) copy number changes of a single TE biopsy alone as evidence for (mosaic) aneuploidy in the embryo remains suboptimal. Integrating genotype information alongside the copy number status could provide a more comprehensive assessment of the embryo's genetic makeup, within and beyond the single TE biopsy. By identifying meiotic aberrations, especially in presumed mosaic embryos, we underscore the potential value of genotyping analysis as a deselection tool, ultimately striving to reduce adverse clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: L.D.W. was supported by the Research Foundation Flanders (FWO; 1S74621N). M.B., K.T., F.V.M., S.J., A.V.T., V.S., D.S., A.D., and S.S. are supported by Ghent University Hospital. B.M. was funded by Ghent University. The authors have no conflicts of interest.

Unveiling the roles of LEMD proteins in cellular processes.

Proteins localized in the inner nuclear membrane (INM) engage in various fundamental cellular processes via their interactions with outer nuclear membrane (ONM) proteins and nuclear lamina. LAP2-emerin-MAN1 domain (LEMD) family proteins, predominantly positioned in the INM, participate in the maintenance of INM functions, including the reconstruction of the nuclear envelope during mitosis, mechanotransduction, and gene transcriptional modulation. Malfunction of LEMD proteins leads to severe tissue-restricted diseases, which may manifest as fatal deformities and defects. In this review, we summarize the significant roles of LEMD proteins in cellular processes, explains the mechanisms of LEMD protein-related diseases, and puts forward questions in less-explored areas like details in tissue-restricted phenotypes. It intends to sort out previous works about LEMD proteins and pave way for future researchers who might discover deeper mechanisms of and better treatment strategies for LEMD protein-related diseases.

Establishment and Characterization of Testis Organoids with Proliferation and Differentiation of Spermatogonial Stem Cells into Spermatocytes and Spermatids.

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.

A comparative study of the cryo-EM structures of Saccharomyces cerevisiae and human anaphase-promoting complex/cyclosome (APC/C).

The anaphase-promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that controls progression through the cell cycle by orchestrating the timely proteolysis of mitotic cyclins and other cell cycle regulatory proteins. Although structures of multiple human APC/C complexes have been extensively studied over the past decade, the Saccharomyces cerevisiae APC/C has been less extensively investigated. Here, we describe medium resolution structures of three S. cerevisiae APC/C complexes: unphosphorylated apo-APC/C and the ternary APC/CCDH1-substrate complex, and phosphorylated apo-APC/C. Whereas the overall architectures of human and S. cerevisiae APC/C are conserved, as well as the mechanism of CDH1 inhibition by CDK-phosphorylation, specific variations exist, including striking differences in the mechanism of coactivator-mediated stimulation of E2 binding, and the activation of APC/CCDC20 by phosphorylation. In contrast to human APC/C in which coactivator induces a conformational change of the catalytic module APC2:APC11 to allow E2 binding, in S. cerevisiae apo-APC/C the catalytic module is already positioned to bind E2. Furthermore, we find no evidence of a phospho-regulatable auto-inhibitory segment of APC1, that in the unphosphorylated human APC/C, sterically blocks the CDC20C-box binding site of APC8. Thus, although the functions of APC/C are conserved from S. cerevisiae to humans, molecular details relating to their regulatory mechanisms differ.

Cohesin ring gates are specialized for meiotic cell division.

Cohesin is a ring complex closed with SMC-1, SMC-3, and a kleisin subunit, mediating sister chromatid cohesion in mitosis and meiosis. Kleisin N- and C-terminal domains interact with SMC-3 and SMC-1, forming two distinct cohesin gates. Whether these gates are specialized for mitosis and meiosis remains elusive. Here, we create Caenorhabditis elegans mutants that express chimeric proteins swapping N- and C-terminal domains between different kleisins to investigate how these gates are specialized for different cell division programs. Replacing the meiotic REC-8 N-terminus with that of a cell division-unrelated kleisin COH-1 or the mitotic kleisin SCC-1 disrupts inter-sister chromatid cohesion and causes severe meiotic defects. Swapping the REC-8 C-terminus with that of COH-1 or SCC-1 largely retains the meiotic functions of REC-8 but causes age-related chromosome abnormalities. A specialized C-terminus is also required for the functions of SCC-1. Furthermore, point mutations in REC-8 C-terminus cause severe meiotic defects without impairing SMC-1-kleisin interaction, suggesting an integrated SMC-1-kleisin gate. These findings suggest the requirements for specialized cohesin gates in different biological processes.

Roles of Histone Acetylation and Deacetylation in Root Development.

Roots are usually underground plant organs, responsible for anchoring to the soil, absorbing water and nutrients, and interacting with the rhizosphere. During root development, roots respond to a variety of environmental signals, contributing to plant survival. Histone post-translational modifications play essential roles in gene expression regulation, contributing to plant responses to environmental cues. Histone acetylation is one of the most studied post-translational modifications, regulating numerous genes involved in various biological processes, including development and stress responses. Although the effect of histone acetylation on plant responses to biotic and abiotic stimuli has been extensively reviewed, no recent reviews exist focusing on root development regulation by histone acetylation. Therefore, this review brings together all the knowledge about the impact of histone acetylation on root development in several plant species, mainly focusing on Arabidopsis thaliana. Here, we summarize the role of histone acetylation and deacetylation in numerous aspects of root development, such as stem cell niche maintenance, cell division, expansion and differentiation, and developmental zone determination. We also emphasize the gaps in current knowledge and propose new perspectives for research toward deeply understanding the role of histone acetylation in root development.

Dynamic phosphorylation of FOXA1 by Aurora B guides post-mitotic gene reactivation.

FOXA1 serves as a crucial pioneer transcription factor during developmental processes and plays a pivotal role as a mitotic bookmarking factor to perpetuate gene expression profiles and maintain cellular identity. During mitosis, the majority of FOXA1 dissociates from specific DNA binding sites and redistributes to non-specific binding sites; however, the regulatory mechanisms governing molecular dynamics and activity of FOXA1 remain elusive. Here, we show that mitotic kinase Aurora B specifies the different DNA binding modes of FOXA1 and guides FOXA1 biomolecular condensation in mitosis. Mechanistically, Aurora B kinase phosphorylates FOXA1 at Serine 221 (S221) to liberate the specific, but not the non-specific, DNA binding. Interestingly, the phosphorylation of S221 attenuates the FOXA1 condensation that requires specific DNA binding. Importantly, perturbation of the dynamic phosphorylation impairs accurate gene reactivation and cell proliferation, suggesting that reversible mitotic protein phosphorylation emerges as a fundamental mechanism for the spatiotemporal control of mitotic bookmarking.

Dynamic Mitotic Localization of the Centrosomal Kinases CDK1, Plk, AurK, and Nek2 in Dictyostelium amoebae.

The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed.

The Role of CDCA2 in tumor genesis, prognosis and future treatments.

The Cell Division Cycle Associated 2 (CDCA2) gene is responsible for encoding a targeting subunit of cell-cycle associated protein. CDCA2 plays a crucial role in various cellular processes, including chromosome segregation and decondensation, nuclear envelope reassembly, microtubule assembly, and DNA damage response. Additionally, CDCA2 is involved in multiple signaling pathways such as the PI3K/Akt pathway and p53 pathway. Undoubtedly, there exists a strong association between CDCA2 and cancer. Numerous studies have reported that elevated levels of CDCA2 are correlated with poor prognosis and several clinicopathological characteristics like tumor size and TNM stage across different types of cancer. Therefore, CDCA2 holds great potential as both a biomarker for diagnosis and a therapeutic target for interventions such as targeted therapies or immunotherapy. Given its promising prospects in scientific research and clinical applications, it is imperative for researchers to delve into the underlying mechanisms of CDCA2 and explore its utilization.

Aurora A Kinase Begins to Localize to the Centrosome in the S-phase of the Cell Cycle in the XL2 Cell Line.

BACKGROUND: The centrosome is one of the principal cell hubs, where numerous proteins important for intracellular regulatory processes are concentrated. One of them, serine-threonine kinase 6, alias Aurora A, is involved in centrosome duplication and mitotic spindle formation and maintenance. METHODS: Long-term vital observations of cells, immunofluorescence analysis of protein localization, synchronization of cells at different phases of the cell cycle, Western blot analysis of protein content were used in the work. RESULTS: In this study, we investigated the dynamics of Aurora A protein accumulation and degradation in the XL2 Xenopus cell line during its 28-hour cell cycle. Using Western blot and immunofluorescence analyses, we demonstrated that Aurora A disappeared from the centrosome within one hour following mitosis and was not redistributed to other cell compartments. Using double Aurora A/Bromodeoxyuridine immunofluorescence labeling of the cells with precisely determined cell cycle stages, we observed that Aurora A reappeared in the centrosome during the S-phase, which was earlier than reported for all other known proteins with mitosis-specific centrosomal localization. Moreover, Aurora A accumulation in the centrosomal region and centrosome separation were asynchronous in the sister cells. CONCLUSIONS: The reported data allowed us to hypothesize that Aurora A is one of the primary links in coordinating centrosome separation and constructing the mitotic spindle.

The 5-Fluorouracil RNA Expression Viewer (5-FUR) Facilitates Interpreting the Effects of Drug Treatment and RRP6 Deletion on the Transcriptional Landscape in Yeast.

Saccharomyces cerevisiae is an excellent model to study the effect of external cues on cell division and stress response. 5-Fluorocuracil (5-FU) has been used to treat solid tumors since several decades. The drug was initially designed to interfere with DNA replication but was later found to exert its antiproliferative effect also via RNA-dependent processes. Since 5-FU inhibits the activity of the 3'-5'-exoribonuclease Rrp6 in yeast and mammals, earlier work has compared the effect of 5-FU treatment and RRP6 deletion at the transcriptome level in diploid synchronized yeast cells. To facilitate interpreting the expression data we have developed an improved 5-Fluorouracil RNA (5-FUR) expression viewer. Users can access information via genome coordinates and systematic or standard names for mRNAs and Xrn1-dependent-, stable-, cryptic-, and meiotic unannotated transcripts (XUTs, SUTs, CUTs, and MUTs). Normalized log2-transformed or linear data can be displayed as filled diagrams, line graphs or color-coded heatmaps. The expression data are useful for researchers interested in processes such as cell cycle regulation, mitotic repression of meiotic genes, the effect of 5-FU treatment and Rrp6 deficiency on the transcriptome and expression profiles of sense/antisense loci that encode overlapping transcripts. The viewer is accessible at http://5fur.genouest.org.

Chromosomal instability increases radiation sensitivity.

Continuous chromosome missegregation over successive mitotic divisions, known as chromosomal instability (CIN), is common in cancer. Increasing CIN above a maximally tolerated threshold leads to cell death due to loss of essential chromosomes. Here, we show in two tissue contexts that otherwise isogenic cancer cells with higher levels of CIN are more sensitive to ionizing radiation, which itself induces CIN. CIN also sensitizes HPV-positive and HPV-negative head and neck cancer patient derived xenograft (PDX) tumors to radiation. Moreover, laryngeal cancers with higher CIN prior to treatment show improved response to radiation therapy. In addition, we reveal a novel mechanism of radiosensitization by docetaxel, a microtubule stabilizing drug commonly used in combination with radiation. Docetaxel causes cell death by inducing CIN due to abnormal multipolar spindles rather than causing mitotic arrest, as previously assumed. Docetaxel-induced CIN, rather than mitotic arrest, is responsible for the enhanced radiation sensitivity observed in vitro and in vivo, challenging the mechanistic dogma of the last 40 years. These results implicate CIN as a potential biomarker and inducer of radiation response, which could provide valuable cancer therapeutic opportunities. Statement of Significance: Cancer cells and laryngeal tumors with higher chromosome missegregation rates are more sensitive to radiation therapy, supporting chromosomal instability as a promising biomarker of radiation response.

LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner.

Amino acid transporters play a vital role in cellular homeostasis by maintaining protein synthesis. L-type amino acid transporter 1 (LAT1/SLC7A5/CD98lc) is a major transporter of large neutral amino acids in cancer cells because of its predominant expression. Although amino acid restriction with various amino acid analog treatments is known to induce mitotic defects, the involvement of amino acid transporters in cell division remains unclear. In this study, we identified that LAT1 is responsible for mitotic progression in a transport activity-independent manner. LAT1 knockdown activates the spindle assembly checkpoint, leading to a delay in metaphase. LAT1 maintains proper spindle orientation with confinement of the lateral cortex localization of the NuMA protein, which mediates the pulling force against the mitotic spindle toward the lateral cortex. Unexpectedly, JPH203, an inhibitor of LAT1 amino acid transport activity, does not affect mitotic progression. Moreover, the transport activity-deficient LAT1 mutant maintains the proper spindle orientation and mitotic progression. LAT1 forms a heterodimer with CD98 (SLC3A2/CD98hc) both in interphase and mitosis. Although CD98 knockdown decreases the plasma membrane localization of LAT1, it does not affect mitotic progression. LAT1 is localized to the Golgi and ER not only at the plasma membrane in interphase, and promotes Golgi unlinking during the mitotic entry, leading to centrosome maturation. These results suggest that LAT1 supports mitotic progression in an amino acid transport activity-independent manner and that Golgi-localized LAT1 is important for mitotic progression through the acceleration of Golgi unlinking and centrosome maturation. These findings reveal a novel LAT1 function in mitosis.

Integrated cytological and transcriptomic analyses provide new insights into restoration of pollen viability in synthetic allotetraploid Brassica carinata.

KEY MESSAGE: Upregulation of genes involved in DNA damage repair and sperm cell differentiation leads to restoration of pollen viability in synthetic allotetraploid B. carinata after chromosome doubling. Apart from the well-known contribution of polyploidy to crop improvement, polyploids can also be induced for other purposes, such as to restore the viability of sterile hybrids. The mechanism related to viability transition between the sterile allodiploid and the fertile allotetraploid after chromosome doubling are not well understood. Here, we synthesised allodiploid B. carinata (2n = 2x = 17) and allotetraploid B. carinata (2n = 4x = 34) as models to investigate the cytological and transcriptomic differences during pollen development. The results showed that after chromosome doubling, the recovery of pollen viability in allotetraploid was mainly reflected in the stabilisation of microtubule spindle morphology, normal meiotic chromosome behaviour, and normal microspore development. Interestingly, the deposition and degradation of synthetic anther tapetum were not affected by polyploidy. Transcription analysis showed that the expression of genes related to DNA repair (DMC1, RAD51, RAD17, SPO11-2), cell cycle differentiation (CYCA1;2, CYCA2;3) and ubiquitination proteasome pathway (UBC4, PIRH2, CDC53) were positively up-regulated during pollen development of synthetic allotetraploid B. carinata. In summary, these results provide some refreshing updates about the ploidy-related restoration of pollen viability in newly synthesised allotetraploid B. carinata.

The Hox Gene, abdominal A controls timely mitotic entry of neural stem cell and their growth during CNS development in Drosophila.

The size of a cell is important for its function and physiology. Interestingly, size variation can be easily observed in clonally derived embryonic and hematopoietic stem cells. Here, we investigated the regulation of stem cell growth and its association with cell fate. We observed heterogeneous sizes of neuroblasts or neural stem cells (NSCs) in the Drosophila ventral nerve cord (VNC). Specifically, thoracic NSCs were larger than those in the abdominal region of the VNC. Our research uncovered a significant role of the Hox gene abdominal A (abdA) in the regulation of abdominal NSC growth. Developmental expression of AbdA retards their growth and delays mitotic entry compared to thoracic NSCs. The targeted loss of abdA enhanced their growth and caused an earlier entry into mitosis with a faster cycling rate. Furthermore, ectopic expression of abdA reduced the size of thoracic NSCs and delayed their entry into mitosis. We suggest that abdA plays an instructive role in regulating NSC size and exit from quiescence. This study demonstrates for the first time the involvement of abdA in NSC fate determination by regulating their growth, entry into mitosis and proliferation rate, and thus their potential to make appropriate number of progeny for CNS patterning.

The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.

The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation.

Mitotic ER-mitochondria contact enhances mitochondrial Ca2+ influx to promote cell division.

Cell division is tightly regulated and requires an expanded energy supply. However, how this energy is generated remains unclear. Here, we establish a correlation between two mitochondrial Ca2+ influx events and ATP production during mitosis. While both events promote ATP production during mitosis, the second event, the Ca2+ influx surge, is substantial. To facilitate this Ca2+ influx surge, the lamin B receptor (LBR) organizes a mitosis-specific endoplasmic reticulum (ER)-mitochondrial contact site (ERMCS), creating a rapid Ca2+ transport pathway. LBR acts as a tether, connecting the ER Ca2+ release channel IP3R with the mitochondrial VDAC2. Depletion of LBR disrupts the Ca2+ influx surge, reduces ATP production, and postpones the metaphase-anaphase transition and subsequent cell division. These findings provide insight into the mechanisms underlying mitotic energy production and supply required for cell proliferation.

A chemical screen identifies PRMT5 as a therapeutic vulnerability for paclitaxel-resistant triple-negative breast cancer.

Paclitaxel-resistant triple negative breast cancer (TNBC) remains one of the most challenging breast cancers to treat. Here, using an epigenetic chemical probe screen, we uncover an acquired vulnerability of paclitaxel-resistant TNBC cells to protein arginine methyltransferases (PRMTs) inhibition. Analysis of cell lines and in-house clinical samples demonstrates that resistant cells evade paclitaxel killing through stabilizing mitotic chromatin assembly. Genetic or pharmacologic inhibition of PRMT5 alters RNA splicing, particularly intron retention of aurora kinases B (AURKB), leading to a decrease in protein expression, and finally results in selective mitosis catastrophe in paclitaxel-resistant cells. In addition, type I PRMT inhibition synergies with PRMT5 inhibition in suppressing tumor growth of drug-resistant cells through augmenting perturbation of AURKB-mediated mitotic signaling pathway. These findings are fully recapitulated in a patient-derived xenograft (PDX) model generated from a paclitaxel-resistant TNBC patient, providing the rationale for targeting PRMTs in paclitaxel-resistant TNBC.