bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2023–09–17
fourteen papers selected by
Valentina Piano, Uniklinik Köln
  1. Analog-sensitive Cdk1 as a tool to study mitotic exit: protein phosphatase 1 is required downstream from Cdk1 inactivation in budding yeast.
  2. A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.
  3. Direct recruitment of Mis18 to interphase spindle pole bodies promotes CENP-A chromatin assembly.
  4. Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.
  5. Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages.
  6. Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment.
  7. Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.
  8. An improved Erk biosensor detects oscillatory Erk dynamics driven by mitotic erasure during early development.
  9. Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning.
  10. Molecular Mechanism of PP2A/B55α Inhibition by IER5.
  11. Centromere: A Trojan horse for genome stability.
  12. CHK1-CDC25A-CDK1 regulate cell cycle progression and protect genome integrity in early mouse embryos.
  13. Mitosis exit followed by death in interphase prevents the development of polyploid giant cancer cells.
  14. High spindle and kinetochore-associated complex subunit-3 expression predicts poor prognosis and correlates with adverse immune infiltration in hepatocellular carcinoma.
  1. Chromosome Res. 2023 Sep 10. 31(3): 27
    Analog-sensitive Cdk1 as a tool to study mitotic exit: protein phosphatase 1 is required downstream from Cdk1 inactivation in budding yeast.
    Jason M Keaton, Benjamin G Workman, Linfeng Xie, James R Paulson.
      We show that specific inactivation of the protein kinase Cdk1/cyclin B (Cdc28/Clb2) triggers exit from mitosis in the budding yeast Saccharomyces cerevisiae. Cells carrying the allele cdc28-as1, which makes Cdk1 (Cdc28) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This caused the cells to leave mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), induction of mating projections ("shmoos") by α-factor, stabilization of Sic1, and degradation of Clb2. It is known that Cdk1 must be inactivated for cells to exit mitosis, but our results show that inactivation of Cdk1 is not only necessary but also sufficient to initiate the transition from mitosis to G1-phase. This result suggests a system in which to test requirements for particular gene products downstream from Cdk1 inactivation, for example, by combining cdc28-as1 with conditional mutations in the genes of interest. Using this approach, we demonstrate that protein phosphatase 1 (PPase1; Glc7 in S. cerevisiae) is required for mitotic exit and reestablishment of interphase following Cdk1 inactivation. This system could be used to test the need for other protein phosphatases downstream from Cdk1 inactivation, such as PPase 2A and Cdc14, and it could be combined with phosphoproteomics to gain information about the substrates that the various phosphatases act upon during mitotic exit.
    Keywords:  1NM-PP1; Cdc28; Cdk1/cyclin B; Cell cycle; MPF; Mitosis; Mitotic exit; Rebudding; cdc28-as1
    DOI:  https://doi.org/10.1007/s10577-023-09736-6
  2. EMBO J. 2023 Sep 15. e112630
    A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.
    Andrea Corno, Marilia H Cordeiro, Lindsey A Allan, Qian-Wei Lim, Elena Harrington, Richard J Smith, Adrian T Saurin.
      Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.
    Keywords:  KNL1; PLK1; PP2A-B56; error-correction signalling; mitotic checkpoint
    DOI:  https://doi.org/10.15252/embj.2022112630
  3. Curr Biol. 2023 Sep 08. pii: S0960-9822(23)01143-0. [Epub ahead of print]
    Direct recruitment of Mis18 to interphase spindle pole bodies promotes CENP-A chromatin assembly.
    Nitobe London, Bethan Medina-Pritchard, Christos Spanos, Juri Rappsilber, A Arockia Jeyaprakash, Robin C Allshire.
      CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP1/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB linker of nucleoskeleton and cytoskeleton (LINC) component Sad1. Mis18C recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1 but operates independently of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.
    Keywords:  CENP-A; Kinetochore; Mis18; S. pombe; Sad1; centromere; chromatin; nucleus; spindle pole
    DOI:  https://doi.org/10.1016/j.cub.2023.08.063
  4. bioRxiv. 2023 Aug 31. pii: 2023.08.31.555365. [Epub ahead of print]
    Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.
    Sathish K R Padi, Margaret R Vos, Rachel J Godek, James R Fuller, Thomas Kruse, Jamin B Hein, Jakob Nilsson, Matthew S Kelker, Rebecca Page, Wolfgang Peti.
      Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. 1 Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases, 2 while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55. 3 While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited. 4 For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent) 6,7 and FAM122A 5 (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.
    DOI:  https://doi.org/10.1101/2023.08.31.555365
  5. Nat Commun. 2023 Sep 13. 14(1): 5652
    Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages.
    Mohammad Zeeshan, Edward Rea, Steven Abel, Kruno Vukušić, Robert Markus, Declan Brady, Antonius Eze, Ravish Rashpa, Aurelia C Balestra, Andrew R Bottrill, Mathieu Brochet, David S Guttery, Iva M Tolić, Anthony A Holder, Karine G Le Roch, Eelco C Tromer, Rita Tewari.
      The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.
    DOI:  https://doi.org/10.1038/s41467-023-41395-3
  6. Cell Rep. 2023 Sep 12. pii: S2211-1247(23)01109-9. [Epub ahead of print] 113098
    Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment.
    Tomohiro Yabushita, Takumi Chinen, Atsuya Nishiyama, Shuhei Asada, Ruka Shimura, Tomoya Isobe, Keita Yamamoto, Naru Sato, Yutaka Enomoto, Yosuke Tanaka, Tomofusa Fukuyama, Hitoshi Satoh, Keiko Kato, Kaori Saitoh, Takamasa Ishikawa, Tomoyoshi Soga, Yasuhito Nannya, Tatsuo Fukagawa, Makoto Nakanishi, Daiju Kitagawa, Toshio Kitamura, Susumu Goyama.
      Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.
    Keywords:  ATR; CHK1; CP: Cancer; DNA hypomethylating agent; DNA methylation; DNMT1; HMA; cholesterol; decitabine; mitosis; mitotic catastrophe
    DOI:  https://doi.org/10.1016/j.celrep.2023.113098
  7. J Cell Biol. 2023 Oct 02. pii: e202212043. [Epub ahead of print]222(10):
    Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.
    Zihan Zhu, Isabelle Becam, Corinne A Tovey, Abir Elfarkouchi, Eugenie C Yen, Fred Bernard, Antoine Guichet, Paul T Conduit.
      Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here, we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.
    DOI:  https://doi.org/10.1083/jcb.202212043
  8. Dev Cell. 2023 Sep 09. pii: S1534-5807(23)00436-7. [Epub ahead of print]
    An improved Erk biosensor detects oscillatory Erk dynamics driven by mitotic erasure during early development.
    Scott G Wilcockson, Luca Guglielmi, Pablo Araguas Rodriguez, Marc Amoyel, Caroline S Hill.
      Extracellular signal-regulated kinase (Erk) signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics in vivo, as a gradient of activated diphosphorylated Erk (P-Erk) is induced by fibroblast growth factor (Fgf) signaling at the blastula margin. Here, we describe an improved Erk-specific biosensor, which we term modified Erk kinase translocation reporter (modErk-KTR). We demonstrate the utility of this biosensor in vitro and in developing zebrafish and Drosophila embryos. Moreover, we show that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Erk activity is rapidly extinguished just prior to mitosis, which we refer to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. Our modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics in vivo.
    Keywords:  Drosophila; Erk; Fgf signaling; kinase translocation reporter; mitosis; zebrafish
    DOI:  https://doi.org/10.1016/j.devcel.2023.08.021
  9. J Cell Biol. 2023 Dec 04. pii: e202301092. [Epub ahead of print]222(12):
    Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning.
    Catherine Sullenberger, Dong Kong, Pegah Avazpour, Delgermaa Luvsanjav, Jadranka Loncarek.
      Centriole duplication is a high-fidelity process driven by Polo-like kinase 4 (Plk4) and a few conserved initiators. Dissecting how Plk4 and its receptors organize within centrosomes is critical to understand the centriole duplication process and biochemical and architectural differences between centrosomes of different species. Here, at nanoscale resolution, we dissect centrosomal localization of Plk4 in G1 and S phase in its catalytically active and inhibited state during centriole duplication and amplification. We build a precise distribution map of Plk4 and its receptor Cep152, as well as Cep44, Cep192, and Cep152-anchoring factors Cep57 and Cep63. We find that Cep57, Cep63, Cep44, and Cep192 localize in ninefold symmetry. However, during centriole maturation, Cep152, which we suggest is the major Plk4 receptor, develops a more complex pattern. We propose that the molecular arrangement of Cep152 creates flexibility for Plk4 and procentriole placement during centriole initiation. As a result, procentrioles form at variable positions in relation to the mother centriole microtubule triplets.
    DOI:  https://doi.org/10.1083/jcb.202301092
  10. bioRxiv. 2023 Aug 29. pii: 2023.08.29.555174. [Epub ahead of print]
    Molecular Mechanism of PP2A/B55α Inhibition by IER5.
    Ruili Cao, Daniel Td Jones, Li Pan, Shumei Wang, Shaun Rawson, Jon C Aster, Stephen C Blacklow.
      PP2A serine/threonine protein phosphatases are heterotrimeric complexes that have a wide range of essential physiologic functions. The B55α form of PP2A has critical roles in cell cycle regulation, mitotic exit, and the DNA damage response 1-6 . Its activity is modulated by additional regulatory proteins, such as ARPP19 7 , FAM122A 8 , and IER5 9 . However, the precise mechanisms underlying the modulation of PP2A activity by these proteins remain elusive. Here, we show that IER5 inhibits pTau dephosphorylation by PP2A/B55α in biochemical assays and report a cryoelectron microscopy structure of the PP2A/B55α-IER5 complex, which reveals that IER5 occludes a surface on B55α used for substrate recruitment 10-12 . Mutation of interface residues on IER5 interferes with recovery of B55α in co-immunoprecipitation assays and suppresses events in squamous carcinoma cells, such as KRT1 expression, that depend on inhibition of PP2A/B55α by IER5 9 . These studies define the molecular basis for PP2A inhibition by IER5 and suggest a roadmap for selective pharmacologic modulation of PP2A/B55α complexes.
    DOI:  https://doi.org/10.1101/2023.08.29.555174
  11. DNA Repair (Amst). 2023 Sep 07. pii: S1568-7864(23)00123-4. [Epub ahead of print]130 103569
    Centromere: A Trojan horse for genome stability.
    Andrea Scelfo, Daniele Fachinetti.
      Centromeres play a key role in the maintenance of genome stability to prevent carcinogenesis and diseases. They are specialized chromosome loci essential to ensure faithful transmission of genomic information across cell generations by mediating the interaction with spindle microtubules. Nonetheless, while fulfilling these essential roles, their distinct repetitive composition and susceptibility to mechanical stresses during cell division render them susceptible to breakage events. In this review, we delve into the present understanding of the underlying causes of centromere fragility, from the mechanisms governing its DNA replication and repair, to the pathways acting to counteract potential challenges. We propose that the centromere represents a "Trojan horse" exerting vital functions that, at the same time, potentially threatens whole genome stability.
    DOI:  https://doi.org/10.1016/j.dnarep.2023.103569
  12. EMBO Rep. 2023 Sep 11. e56530
    CHK1-CDC25A-CDK1 regulate cell cycle progression and protect genome integrity in early mouse embryos.
    Lucie Knoblochova, Tomas Duricek, Michaela Vaskovicova, Chrysoula Zorzompokou, Diana Rayova, Ivana Ferencova, Vladimir Baran, Richard M Schultz, Eva R Hoffmann, David Drutovic.
      After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated and transcriptionally quiescent cells into early cleavage-stage blastomeres that are transcriptionally active and totipotent. This developmental transition is accompanied by cell cycle adaptation, such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in two-cell stage mouse embryos. Finally, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.
    Keywords:  CDC25A phosphatase; CDK1 kinase; CHK1 kinase; cell cycle regulation; early mouse embryos
    DOI:  https://doi.org/10.15252/embr.202256530
  13. bioRxiv. 2023 Sep 01. pii: 2023.08.31.555795. [Epub ahead of print]
    Mitosis exit followed by death in interphase prevents the development of polyploid giant cancer cells.
    Juan Jesus Vicente, Kainat Khan, Grant Tillinghast, José L McFaline-Figueroa, Yasemin Sancak, Nephi Stella.
      Microtubule targeting agents ( MTAs ) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells can escape death in mitosis and exit mitosis, and become malignant polyploid giant cancer cells ( PGCC ). Considering the low number of malignant cells undergoing mitosis in tumor tissue, killing these cells in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule assembly, preferentially kills cancer cells in interphase as opposed to mitosis, and avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces an integrated stress response and promotes mitochondria fission accompanied by a reduction in energy metabolism. This cell response may underly death in interphase and avoid the development of PGCC.
    DOI:  https://doi.org/10.1101/2023.08.31.555795
  14. World J Gastrointest Surg. 2023 Aug 27. 15(8): 1600-1614
    High spindle and kinetochore-associated complex subunit-3 expression predicts poor prognosis and correlates with adverse immune infiltration in hepatocellular carcinoma.
    Lin-Lin Zheng, Ya-Ru Wang, Zhen-Rong Liu, Zhi-Hao Wang, Chang-Cheng Tao, Yong-Gang Xiao, Kai Zhang, An-Ke Wu, Hai-Yang Li, Jian-Xiong Wu, Ting Xiao, Wei-Qi Rong.
       BACKGROUND: Spindle and kinetochore-associated complex subunit 3 (SKA3) is a malignancy-associated gene that plays a critical role in the regulation of chromosome separation and cell division. However, the molecular mechanism through which SKA3 regulates tumor cell proliferation in hepatocellular carcinoma (HCC) has not been fully elucidated.
    AIM: To investigate the molecular mechanisms underlying the role of SKA3 in HCC.
    METHODS: SKA3 expression, clinicopathological, and survival analyses were performed using multiple public database platforms, and the results were verified by Western blot and immunohistochemistry staining using collected clinical samples. Functional enrichment analyses were performed to evaluate the biological functions and molecular mechanisms of SKA3 in HCC. Furthermore, the Tumor Immune Estimation Resource and single-sample Gene Set Enrichment Analysis (ssGSEA) algorithms were utilized to investigate the abundance of tumor-infiltrating immune cells in HCC. The response to chemotherapeutic drugs was evaluated by the R package "pRRophetic".
    RESULTS: We found that upregulated SKA3 expression was significantly correlated with poor prognosis in patients with HCC. Multivariable Cox regression analysis indicated that SKA3 was an independent risk factor for survival. GSEA revealed that SKA3 expression may facilitate proliferation and migratory processes by regulating the cell cycle and DNA repair. Moreover, patients with high SKA3 expression had significantly decreased ratios of CD8+ T cells, natural killer cells, and dendritic cells. Drug sensitivity analysis showed that the high SKA3 group was more sensitive to sorafenib, sunitinib, paclitaxel, doxorubicin, gemcitabine, and vx-680.
    CONCLUSION: High SKA3 expression led to poor prognosis in patients with HCC by enhancing HCC proliferation and repressing immune cell infiltration surrounding HCC. SKA3 may be used as a biomarker for poor prognosis and as a therapeutic target in HCC.
    Keywords:  Hepatocellular carcinoma; Immune infiltration cells; Prognosis; Spindle and kinetochore-associated protein 3
    DOI:  https://doi.org/10.4240/wjgs.v15.i8.1600
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