bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2023–01–29
ten papers selected by
Valentina Piano, Uniklinik Köln
  1. CCDC86 is a novel Ki-67-interacting protein important for cell division.
  2. Nanoscale structural organization and stoichiometry of the budding yeast kinetochore.
  3. USP49 deubiquitinase regulates the mitotic spindle checkpoint and prevents aneuploidy.
  4. Master mitotic kinases regulate viral genome delivery during papillomavirus cell entry.
  5. Genomic instability caused by Arp2/3 complex inactivation results in micronucleus biogenesis and cellular senescence.
  6. Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color SMLM imaging.
  7. Phosphorylation of the prolyl isomerase Cyclophilin A regulates its localisation and release from the centrosome during mitosis.
  8. Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis.
  9. Dynamics and regulation of mitotic chromatin accessibility bookmarking at single-cell resolution.
  10. A nucleotide binding-independent role for γ-tubulin in microtubule capping and cell division.
  1. J Cell Sci. 2023 Jan 15. pii: jcs260391. [Epub ahead of print]136(2):
    CCDC86 is a novel Ki-67-interacting protein important for cell division.
    Konstantinos Stamatiou, Aldona Chmielewska, Shinya Ohta, William C Earnshaw, Paola Vagnarelli.
      The chromosome periphery is a network of proteins and RNAs that coats the outer surface of mitotic chromosomes. Despite the identification of new components, the functions of this complex compartment are poorly characterised. In this study, we identified a novel chromosome periphery-associated protein, CCDC86 (also known as cyclon). Using a combination of RNA interference, microscopy and biochemistry, we studied the functions of CCDC86 in mitosis. CCDC86 depletion resulted in partial disorganisation of the chromosome periphery with alterations in the localisation of Ki-67 (also known as MKI67) and nucleolin (NCL), and the formation of abnormal cytoplasmic aggregates. Furthermore, CCDC86-depleted cells displayed errors in chromosome alignment, altered spindle length and increased apoptosis. These results suggest that, within the chromosome periphery, different subcomplexes that include CCDC86, nucleolin and B23 (nucleophosmin or NPM1) are required for mitotic spindle regulation and correct kinetochore-microtubule attachments, thus contributing to chromosome segregation in mitosis. Moreover, we identified CCDC86 as a MYCN-regulated gene, the expression levels of which represent a powerful marker for prognostic outcomes in neuroblastoma.
    Keywords:  CCDC86; Cell division; Chromosomes; Ki-67; MYCN
    DOI:  https://doi.org/10.1242/jcs.260391
  2. J Cell Biol. 2023 Apr 03. pii: e202209094. [Epub ahead of print]222(4):
    Nanoscale structural organization and stoichiometry of the budding yeast kinetochore.
    Konstanty Cieslinski, Yu-Le Wu, Lisa Nechyporenko, Sarah Janice Hörner, Duccio Conti, Michal Skruzny, Jonas Ries.
      Proper chromosome segregation is crucial for cell division. In eukaryotes, this is achieved by the kinetochore, an evolutionarily conserved multiprotein complex that physically links the DNA to spindle microtubules and takes an active role in monitoring and correcting erroneous spindle-chromosome attachments. Our mechanistic understanding of these functions and how they ensure an error-free outcome of mitosis is still limited, partly because we lack a complete understanding of the kinetochore structure in the cell. In this study, we use single-molecule localization microscopy to visualize individual kinetochore complexes in situ in budding yeast. For major kinetochore proteins, we measured their abundance and position within the metaphase kinetochore. Based on this comprehensive dataset, we propose a quantitative model of the budding yeast kinetochore. While confirming many aspects of previous reports based on bulk imaging, our results present a unifying nanoscale model of the kinetochore in budding yeast.
    DOI:  https://doi.org/10.1083/jcb.202209094
  3. Cell Death Dis. 2023 Jan 26. 14(1): 60
    USP49 deubiquitinase regulates the mitotic spindle checkpoint and prevents aneuploidy.
    Diana Campos-Iglesias, Julia M Fraile, Gabriel Bretones, Alejandro A Montero, Elena Bonzon-Kulichenko, Jesús Vázquez, Carlos López-Otín, José M P Freije.
      The spindle assembly checkpoint (SAC) is an essential mechanism that ensures the accurate chromosome segregation during mitosis, thus preventing genomic instability. Deubiquitinases have emerged as key regulators of the SAC, mainly by determining the fate of proteins during cell cycle progression. Here, we identify USP49 deubiquitinase as a novel regulator of the spindle checkpoint. We show that loss of USP49 in different cancer cell lines impairs proliferation and increases aneuploidy. In addition, USP49-depleted cells overcome the arrest induced by the SAC in the presence of nocodazole. Finally, we report new binding partners of USP49, including ribophorin 1, USP44, and different centrins.
    DOI:  https://doi.org/10.1038/s41419-023-05600-x
  4. Nat Commun. 2023 Jan 23. 14(1): 355
    Master mitotic kinases regulate viral genome delivery during papillomavirus cell entry.
    Matteo Rizzato, Fuxiang Mao, Florian Chardon, Kun-Yi Lai, Ruth Villalonga-Planells, Hannes C A Drexler, Marion E Pesenti, Mert Fiskin, Nora Roos, Kelly M King, Shuaizhi Li, Eduardo R Gamez, Lilo Greune, Petra Dersch, Claudia Simon, Murielle Masson, Koenraad Van Doorslaer, Samuel K Campos, Mario Schelhaas.
      Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2's chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.
    DOI:  https://doi.org/10.1038/s41467-023-35874-w
  5. PLoS Genet. 2023 Jan 27. 19(1): e1010045
    Genomic instability caused by Arp2/3 complex inactivation results in micronucleus biogenesis and cellular senescence.
    Elena L Haarer, Corey J Theodore, Shirley Guo, Ryan B Frier, Kenneth G Campellone.
      The Arp2/3 complex is an actin nucleator with well-characterized activities in cell morphogenesis and movement, but its roles in nuclear processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity and cell cycle progression using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 complex ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Micronuclei arise in ArpC2 iKO cells due to chromatin segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged DNA fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. In the nuclei of Arp2/3-depleted cells, the tumor suppressor p53 is activated and the cell cycle inhibitor Cdkn1a/p21 mediates a G1 arrest. In the cytosol, micronuclei are recognized by the DNA sensor cGAS, which is important for stimulating a STING- and IRF3-associated interferon response. These studies establish functional requirements for the mammalian Arp2/3 complex in mitotic spindle organization and genome stability. They also expand our understanding of the mechanisms leading to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.
    DOI:  https://doi.org/10.1371/journal.pgen.1010045
  6. J Cell Biol. 2023 Apr 03. pii: e202209096. [Epub ahead of print]222(4):
    Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color SMLM imaging.
    David Virant, Ilijana Vojnovic, Jannik Winkelmeier, Marc Endesfelder, Bartosz Turkowyd, David Lando, Ulrike Endesfelder.
      The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.
    DOI:  https://doi.org/10.1083/jcb.202209096
  7. Cell Cycle. 2023 Jan 23. 1-16
    Phosphorylation of the prolyl isomerase Cyclophilin A regulates its localisation and release from the centrosome during mitosis.
    Rebecca Gorry, Kieran Brennan, Paul Tm Lavin, Rebecca Sheridan, Margaret M Mc Gee.
      The centrosome acts as a protein platform from which proteins are deployed to function throughout the cell cycle. Previously, we have shown that the prolyl isomerase Cyclophilin A (CypA) localizes to the centrosome in interphase and re-localizes to the midbody during mitosis where it functions in cytokinesis. In this study, investigation of CypA by SDS-PAGE during the cell cycle reveals that it undergoes a mobility shift during mitosis, indicative of a post-translational modification, which may correlate with its subcellular re-localization. Due to the lack of a phospho-specific antibody, we used site-directed mutagenesis to demonstrate that the previously identified serine 77 phosphorylation site within CypA is important for control of CypA centrosome localization. Furthermore, CypA is shown to interact with the mitotic NIMA-related kinase 2 (Nek2) during interphase and mitosis, while also interacting with the Nek2-antagonist PP1 during interphase but not during mitosis, suggesting a potential role for the Nek2-PP1 complex in CypA phospho-regulation. In support of this, Nek2 is capable of phosphorylating CypA in vitro. Overall, this work reveals that phosphorylation of CypA at serine 77 is important for its release from the centrosome during mitosis and may be regulated by the activity of Nek2 and PP1 during the cell cycle.
    Keywords:  Cyclophilin A; Nek2; centrosome; midbody; mitosis; phosphorylation
    DOI:  https://doi.org/10.1080/15384101.2023.2167430
  8. J Cell Sci. 2023 Jan 25. pii: jcs.260568. [Epub ahead of print]
    Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis.
    Sherman Foo, Amaury Cazenave-Gassiot, Markus R Wenk, Snezhana Oliferenko.
      Nuclear envelope (NE) expansion must be controlled to maintain nuclear shape and function. The nuclear membrane expands massively during 'closed' mitosis, enabling chromosome segregation within an intact NE. Phosphatidic acid (PA) and diacylglycerol (DG) can both serve as biosynthetic precursors for membrane lipid synthesis. How they are regulated in time and space and what are the implications of changes in their flux for mitotic fidelity is largely unknown. Using genetically encoded PA and DG probes, we show that DG is depleted from the inner nuclear membrane during mitosis in the fission yeast Schizosaccharomyces pombe, but PA does not accumulate, indicating that it is rerouted to membrane synthesis. We demonstrate that DG-to-PA conversion catalysed by the diacylglycerol kinase Dgk1 and direct glycerophospholipid synthesis from DG by diacylglycerol cholinephosphotransferase / ethanolaminephosphotransferase Ept1 reinforce NE expansion. We conclude that DG consumption through both de novo and the Kennedy pathways fuels a spike in glycerophospholipid biosynthesis, controlling NE expansion, and ultimately, mitotic fidelity.
    Keywords:  Closed mitosis; Diacylglycerol; Nuclear envelope
    DOI:  https://doi.org/10.1242/jcs.260568
  9. Sci Adv. 2023 Jan 25. 9(4): eadd2175
    Dynamics and regulation of mitotic chromatin accessibility bookmarking at single-cell resolution.
    Qiaoni Yu, Xu Liu, Jingwen Fang, Huihui Wu, Chuang Guo, Wen Zhang, Nianping Liu, Chen Jiang, Qing Sha, Xiao Yuan, Zhikai Wang, Kun Qu.
      Although mitotic chromosomes are highly compacted and transcriptionally inert, some active chromatin features are retained during mitosis to ensure the proper postmitotic reestablishment of maternal transcriptional programs, a phenomenon termed "mitotic bookmarking." However, the dynamics and regulation of mitotic bookmarking have not been systemically surveyed. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we examined 6538 mitotic L02 human liver cells of variable stages and found that chromatin accessibility remained changing throughout cell division, with a constant decrease until metaphase and a gradual increase as chromosomes segregated. In particular, a subset of chromatin regions were identified to remain open throughout mitosis, and genes associated with these bookmarked regions are primarily linked to rapid reactivation upon mitotic exit. We also demonstrated that nuclear transcription factor Y subunit α (NF-YA) preferentially occupied bookmarked regions and contributed to transcriptional reactivation after mitosis. Our study uncovers the dynamic and regulatory blueprint of mitotic bookmarking.
    DOI:  https://doi.org/10.1126/sciadv.add2175
  10. J Cell Biol. 2023 Mar 06. pii: e202204102. [Epub ahead of print]222(3):
    A nucleotide binding-independent role for γ-tubulin in microtubule capping and cell division.
    Adi Y Berman, Michal Wieczorek, Amol Aher, Paul Dominic B Olinares, Brian T Chait, Tarun M Kapoor.
      The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human γ-TuRC's capping activity. Single filament assays showed that the γ-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of ∼1 min. Reconstituted γ-TuRCs with nucleotide-binding deficient γ-tubulin (γ-tubulinΔGTP) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of γ-tubulin suppressed non-centrosomal microtubule formation, add-back of γ-tubulinΔGTP could substantially restore this process. Our results suggest that γ-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division.
    DOI:  https://doi.org/10.1083/jcb.202204102
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