bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2024‒07‒14
sixteen papers selected by
Valentina Piano, Uniklinik Köln



  1. Proc Natl Acad Sci U S A. 2024 Jul 16. 121(29): e2321647121
      Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation.
    Keywords:  actin; microtubules; mitosis; mitotic spindle; protein kinase
    DOI:  https://doi.org/10.1073/pnas.2321647121
  2. Nat Commun. 2024 Jul 10. 15(1): 5794
      Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.
    DOI:  https://doi.org/10.1038/s41467-024-50167-6
  3. Curr Opin Cell Biol. 2024 Jul 08. pii: S0955-0674(24)00075-9. [Epub ahead of print]89 102396
      Centromeres are specialized chromosomal domains where the kinetochores assemble during cell division to ensure accurate transmission of the genetic information to the two daughter cells. The centromeric function is evolutionary conserved and, in most organisms, centromeres are epigenetically defined by a unique chromatin containing the histone H3 variant CENP-A. The canonical regulators of CENP-A assembly and maintenance are well-known, yet some of the molecular mechanisms regulating this complex process have only recently been unveiled. We review the most recent advances on the topic, including the emergence of new and unexpected factors that favor and regulate CENP-A assembly and/or maintenance.
    DOI:  https://doi.org/10.1016/j.ceb.2024.102396
  4. Cell Rep. 2024 Jul 08. pii: S2211-1247(24)00748-4. [Epub ahead of print]43(7): 114419
      The compaction of chromatin into mitotic chromosomes is essential for faithful transmission of the genome during cell division. In eukaryotes, chromosome morphogenesis is regulated by the condensin complex, though the exact mechanism used to target condensin to chromatin and initiate condensation is not understood. Here, we reveal that condensin contains an intrinsically disordered region (IDR) that modulates its association with chromatin in early mitosis and exhibits phase separation. We describe DNA-binding motifs within the IDR that, upon deletion, inflict striking defects in chromosome condensation and segregation, ill-timed condensin turnover on chromatin, and cell death. Importantly, we demonstrate that the condensin IDR can impart cell cycle regulatory functions when transferred to other subunits within the complex, indicating its autonomous nature. Collectively, our study unveils the molecular basis for the initiation of chromosome condensation in early mitosis and how this process ultimately promotes genomic stability and faultless cell division.
    Keywords:  CP: Molecular biology; IDR; Smc4; chromatin; chromosome condensation; condensin complex; genomic stability; intrinsically disordered protein region; mitosis; phase separation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114419
  5. Proc Natl Acad Sci U S A. 2024 Jul 16. 121(29): e2404551121
      Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.
    Keywords:  aneuploidy; chromosome segregation; mechanical stress; mitosis; mitotic spindle
    DOI:  https://doi.org/10.1073/pnas.2404551121
  6. bioRxiv. 2024 Jun 24. pii: 2024.06.24.600220. [Epub ahead of print]
      Moderating the pool of active ribosomal subunits is critical for maintaining global translation rates. A factor critical for modulating the pool of 60S ribosomal subunits is eukaryotic translation initiation factor-6 (eIF6). Release of eIF6 from the 60S subunit is important for permitting 60S interactions with 40S to form the 80S monosome. Here, using a mutant of eIF6 (N106S) that interacts poorly with the 60S subunit, we show that the ribosomal subunit associations are deregulated in the absence of eIF6 and lead to an increase in empty 80S ribosomes that decreases global protein synthesis rates. Intriguingly, altering 80S ribosome availability markedly affects the mitotic phase and leads to chromosome segregation defects that induces delays in mitotic exit and mitotic catastrophe. Ribo-Seq analysis of the eIF6-N106S mutant shows a significant downregulation in the translation efficiencies of genes associated with mitosis and cytoskeleton, and specifically transcripts with long 3'UTRs. eIF6-N106S mutation also markedly affects cancer invasion, and this role is correlated with the overexpression of eIF6 only in high-grade invasive bladder and breast cancers. Thus, this study highlights the importance of therapeutically targeting the eIF6-60S interaction interface in cancers and the importance of modulating active 80S ribosome availability for mitotic translation and mitotic exit.
    DOI:  https://doi.org/10.1101/2024.06.24.600220
  7. J Cell Sci. 2024 Jul 11. pii: jcs.261928. [Epub ahead of print]
      The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions of the cell. Homo/heterodimers of these protein homologs, majorly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or survive with severe growth defects showing gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work we investigated their centromere/kinetochore-related functions. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and unshared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins, and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.
    Keywords:  14-3-3 proteins; Bmh1; Bmh2; Budding yeast; Chromosome congression; Chromosome segregation; Kinetochore
    DOI:  https://doi.org/10.1242/jcs.261928
  8. bioRxiv. 2024 Jun 28. pii: 2024.06.26.600663. [Epub ahead of print]
      Eukaryotic genomes are organized by condensin into 3D chromosomal architectures suitable for chromosomal segregation during mitosis. However, molecular mechanisms underlying the condensin-mediated chromosomal organization remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits in fission yeast, Schizosaccharomyces pombe. We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensinmediated chromatin domains during mitosis and causes chromosomal segregation defects. The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains. Furthermore, this study predicts that mediator-driven transcription of mitotically activated genes contributes to forming domain boundaries via phase separation. This study provides a novel insight into how genome-wide gene expression during mitosis is transformed into the functional chromosomal architecture suitable for chromosomal segregation.
    DOI:  https://doi.org/10.1101/2024.06.26.600663
  9. bioRxiv. 2024 Jun 27. pii: 2024.06.27.600994. [Epub ahead of print]
      Mammalian centromeres are satellite-rich chromatin domains that serve as sites for kinetochore complex assembly. Centromeres are highly variable in sequence and satellite organization across species, but the processes that govern the co-evolutionary dynamics between rapidly evolving centromeres and their associated kinetochore proteins remain poorly understood. Here, we pursue a course of phylogenetic analyses to investigate the molecular evolution of the complete kinetochore complex across primate and rodent species with divergent centromere repeat sequences and features. We show that many protein components of the core centromere associated network (CCAN) harbor signals of adaptive evolution, consistent with their intimate association with centromere satellite DNA and roles in the stability and recruitment of additional kinetochore proteins. Surprisingly, CCAN and outer kinetochore proteins exhibit comparable rates of adaptive divergence, suggesting that changes in centromere DNA can ripple across the kinetochore to drive adaptive protein evolution within distant domains of the complex. Our work further identifies kinetochore proteins subject to lineage-specific adaptive evolution, including rapidly evolving proteins in species with centromere satellites characterized by higher-order repeat structure and lacking CENP-B boxes. Thus, features of centromeric chromatin beyond the linear DNA sequence may drive selection on kinetochore proteins. Overall, our work spotlights adaptively evolving proteins with diverse centromere-associated functions, including centromere chromatin structure, kinetochore protein assembly, kinetochore-microtubule association, cohesion maintenance, and DNA damage response pathways. These adaptively evolving kinetochore protein candidates present compelling opportunities for future functional investigations exploring how their concerted changes with centromere DNA ensure the maintenance of genome stability.
    DOI:  https://doi.org/10.1101/2024.06.27.600994
  10. iScience. 2024 Jul 19. 27(7): 110151
      Point mutations in KIF22 have been linked to spondyloepimetaphyseal dysplasia with joint laxity, type 2 (SEMDJL2). Skeletal features of SEMDJL2 include short stature and joint laxity. Mechanisms underlying these limb abnormalities are unknown. Here in this manuscript, we have investigated the function of KIF22 in chondrocytes. Quantitative PCR and immunostaining revealed that Kif22 was highly expressed in proliferating-zone growth-plate chondrocytes. Kif22 knockdown resulted in defective mitotic spindle formation and reduced cell proliferation. Forced expression of SEMDJL-associated mutant Kif22 constructs likewise induced abnormal mitotic spindle morphology and reduced proliferation. Mice expressing a KIF22 truncation mutant had shorter growth plates and shorter tibial bones compared to wild-type mice. These results suggest that KIF22 regulates mitotic spindle formation in proliferating chondrocytes thereby linking the stunted longitudinal bone growth observed in SEMDJL2 to failures of chondrocyte division.
    Keywords:  Molecular biology; cell biology
    DOI:  https://doi.org/10.1016/j.isci.2024.110151
  11. Genetics. 2024 Jul 10. pii: iyae108. [Epub ahead of print]
      Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to non-centromeric regions contributes to chromosomal instability (CIN) in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers is associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box (SCF) ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and CIN under normal physiological conditions. Mck1-mediated phosphorylation of SCF-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4 and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus site (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, CIN, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the CIN phenotype of GAL-cse4-3A strain, suggesting that the CIN phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and CIN. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.
    Keywords:  CENP-A; Cdc4; Cse4; Mck1; centromere
    DOI:  https://doi.org/10.1093/genetics/iyae108
  12. Signal Transduct Target Ther. 2024 Jul 12. 9(1): 181
      Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.
    DOI:  https://doi.org/10.1038/s41392-024-01896-z
  13. bioRxiv. 2024 Jun 29. pii: 2024.06.28.601230. [Epub ahead of print]
      Protein phosphorylation regulates many steps in the cell division process including cytokinesis. In the fission yeast S. pombe, the anillin-like protein Mid1 sets the cell division plane and is regulated by phosphorylation. Multiple protein kinases act on Mid1, but no protein phosphatases have been shown to regulate Mid1. Here, we discovered that the conserved protein phosphatase PP2A-B56 is required for proper cytokinesis by promoting Mid1 protein levels. We find that par1Δ cells lacking the primary B56 subunit divide asymmetrically due to the assembly of misplaced cytokinetic rings that slide toward cell tips. These par1Δ mutants have reduced whole-cell levels of Mid1 protein, leading to reduced Mid1 at the cytokinetic ring. Restoring proper Mid1 expression suppresses par1Δ cytokinesis defects. This work identifies a new PP2A-B56 pathway regulating cytokinesis through Mid1, with implications for control of cytokinesis in other organisms.
    DOI:  https://doi.org/10.1101/2024.06.28.601230
  14. bioRxiv. 2024 Jun 29. pii: 2024.06.27.601054. [Epub ahead of print]
      Cytokinesis, the physical division of one cell into two, is typically assumed to use the same molecular process across animal cells. However, regulation of cell division can vary significantly among different cell types, even within the same multicellular organism. Using six fast-acting temperature-sensitive (ts) cytokinesis-defective mutants, we found that each had unique cell type-specific profiles in the early C. elegans embryo. Certain cell types were more sensitive than others to actomyosin and spindle signaling disruptions, disrupting two members of the same complex could result in different phenotypes, and protection against actomyosin inhibition did not always protect against spindle signaling inhibition.
    DOI:  https://doi.org/10.1101/2024.06.27.601054
  15. Nat Commun. 2024 Jul 10. 15(1): 5776
      The Ser/Thr protein phosphatase 2 A (PP2A) regulates the dephosphorylation of many phosphoproteins. Substrate recognition are mediated by B regulatory subunits. Here, we report the identification of a substrate conserved motif [RK]-V-x-x-[VI]-R in FAM122A, an inhibitor of B55α/PP2A. This motif is necessary for FAM122A binding to B55α, and computational structure prediction suggests the motif, which is helical, blocks substrate docking to the same site. In this model, FAM122A also spatially constrains substrate access by occluding the catalytic subunit. Consistently, FAM122A functions as a competitive inhibitor as it prevents substrate binding and dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. FAM122A deficiency in human cell lines reduces the proliferation rate, cell cycle progression, and hinders G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells attenuates CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a short helical motif (SHeM)-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.
    DOI:  https://doi.org/10.1038/s41467-024-50015-7
  16. Sci Rep. 2024 Jul 10. 14(1): 15912
      Pancreatic adenocarcinoma is one of the most aggressive and lethal forms of cancer. Chemotherapy is the primary treatment for pancreatic cancer, but resistance to the drugs used remains a major challenge. A genome-wide CRISPR interference and knockout screen in the PANC-1 cell line with the drug nab-paclitaxel has identified a group of spindle assembly checkpoint (SAC) genes that enhance survival in nab-paclitaxel. Knockdown of these SAC genes (BUB1B, BUB3, and TTK) attenuates paclitaxel-induced cell death. Cells treated with the small molecule inhibitors BAY 1217389 or MPI 0479605, targeting the threonine tyrosine kinase (TTK), also enhance survival in paclitaxel. Overexpression of these SAC genes does not affect sensitivity to paclitaxel. These discoveries have helped to elucidate the mechanisms behind paclitaxel cytotoxicity. The outcomes of this investigation may pave the way for a deeper comprehension of the diverse responses of pancreatic cancer to therapies including paclitaxel. Additionally, they could facilitate the formulation of novel treatment approaches for pancreatic cancer.
    DOI:  https://doi.org/10.1038/s41598-024-66244-1