bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022–08–28
seven papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology
  1. Role of phosphorylation of Cdc20 in the regulation of the action of APC/C in mitosis.
  2. Kinesin-14 HSET may not oppose kinesin-5 Eg5 activity in RPE-1 cells.
  3. Composition and organization of kinetochores show plasticity in apicomplexan chromosome segregation.
  4. Analysis of the potential role of fission yeast PP2A in spindle assembly checkpoint inactivation.
  5. Lysosome exocytosis is required for mitosis in mammalian cells.
  6. Cell Cycle Regulation by Integrin-Mediated Adhesion.
  7. Polo-like kinase-1, Aurora kinase A and WEE1 kinase are promising druggable targets in CML cells displaying BCR::ABL1-independent resistance to tyrosine kinase inhibitors.
  1. Proc Natl Acad Sci U S A. 2022 Aug 30. 119(35): e2210367119
    Role of phosphorylation of Cdc20 in the regulation of the action of APC/C in mitosis.
    Danielle Shevah-Sitry, Shirly Miniowitz-Shemtov, Adar Teichner, Sharon Kaisari, Avram Hershko.
      The ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for the control of mitosis, and its activity is subject to tight regulation. In early mitosis, APC/C is inhibited by the mitotic checkpoint system, but subsequently it regains activity and promotes metaphase-anaphase transition by targeting cyclin B and securin for degradation. The phosphorylation of APC/C by the mitotic protein kinase Cdk1-cyclin B facilitates its interaction with its coactivator Cdc20, while the phosphorylation of Cdc20 inhibits its binding to APC/C. This raises the question of how Cdc20 binds to APC/C under conditions of high Cdk1 activity. It seemed possible that the opposing action of protein phosphatases produces a fraction of unphosphorylated Cdc20 that binds to APC/C. We found, however, that while inhibitors of protein phosphatases PP2A and PP1 increased the overall phosphorylation of Cdc20 in anaphase extracts from Xenopus eggs, they did not decrease the levels of Cdc20 bound to APC/C. Searching for alternative mechanisms, we found that following the binding of Cdc20 to APC/C, it became significantly protected against phosphorylation by Cdk1. Protection was mainly at threonine sites at the N-terminal region of Cdc20, known to affect its interaction with APC/C. A model is proposed according to which a pool of unphosphorylated Cdc20, originating from initial stages of mitosis or from phosphatase action, combines with phosphorylated APC/C and thus becomes stabilized against further phosphorylation, possibly by steric hindrance of Cdk1 action. This pool of APCCdc20 appears to be required for the regulation of APC/C activity at different stages of mitosis.
    Keywords:  cell cycle; mitosis; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2210367119
  2. MicroPubl Biol. 2022 ;2022
    Kinesin-14 HSET may not oppose kinesin-5 Eg5 activity in RPE-1 cells.
    Frederique Carlier-Grynkorn, Daniele Fachinetti, Phong T Tran.
      Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines.
    DOI:  https://doi.org/10.17912/micropub.biology.000623
  3. J Cell Biol. 2022 Sep 05. pii: e202111084. [Epub ahead of print]221(9):
    Composition and organization of kinetochores show plasticity in apicomplexan chromosome segregation.
    Lorenzo Brusini, Nicolas Dos Santos Pacheco, Eelco C Tromer, Dominique Soldati-Favre, Mathieu Brochet.
      Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify 13 previously unknown components of kinetochores in Apicomplexa. Apicomplexan kinetochores are highly divergent in sequence and composition from animal and fungal models. The nanoscale organization includes at least four discrete compartments, each displaying different biochemical interactions, subkinetochore localizations and evolutionary rates across the phylum. We reveal alignment of kinetochores at the metaphase plate in both Plasmodium berghei and Toxoplasma gondii, suggestive of a conserved "hold signal" that prevents precocious entry into anaphase. Finally, we show unexpected plasticity in kinetochore composition and segregation between apicomplexan lifecycle stages, suggestive of diverse requirements to maintain fidelity of chromosome segregation across parasite modes of division.
    DOI:  https://doi.org/10.1083/jcb.202111084
  4. FASEB J. 2022 Sep;36(9): e22524
    Analysis of the potential role of fission yeast PP2A in spindle assembly checkpoint inactivation.
    Da-Jie Deng, Xi Wang, Kai-Ye Yue, Yamei Wang, Quan-Wen Jin.
      As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation.
    Keywords:  PP2A; fission yeast; spindle checkpoint inactivation
    DOI:  https://doi.org/10.1096/fj.202101884R
  5. Biochem Biophys Res Commun. 2022 Aug 16. pii: S0006-291X(22)01137-8. [Epub ahead of print]626 211-219
    Lysosome exocytosis is required for mitosis in mammalian cells.
    Charlotte Nugues, Dayani Rajamanoharan, Robert D Burgoyne, Lee P Haynes, Nordine Helassa.
      Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. In this paper we demonstrate how lysosomes dynamically redistribute during mitosis and exocytose. Antagonism of lysosomal exocytosis by pharmacological and genetic approaches causes mitosis failure in a significant proportion of cells. We speculate that either lysosomal membrane or luminal content release, possibly both, are therefore required for normal mitosis progression. These findings are important as they reveal a new process required for successful cell division.
    Keywords:  Exocytosis; Lysosome; Mitosis; Phosphoinositide; TIRFM
    DOI:  https://doi.org/10.1016/j.bbrc.2022.08.024
  6. Cells. 2022 Aug 14. pii: 2521. [Epub ahead of print]11(16):
    Cell Cycle Regulation by Integrin-Mediated Adhesion.
    Siamak A Kamranvar, Bhavna Rani, Staffan Johansson.
      Cell cycle and cell adhesion are two interdependent cellular processes regulating each other, reciprocally, in every cell cycle phase. The cell adhesion to the extracellular matrix (ECM) via integrin receptors triggers signaling pathways required for the cell cycle progression; the passage from the G1 to S phase and the completion of cytokinesis are the best-understood events. Growing evidence, however, suggests more adhesion-dependent regulatory aspects of the cell cycle, particularly during G2 to M transition and early mitosis. Conversely, the cell cycle machinery regulates cell adhesion in manners recently shown driven mainly by cyclin-dependent kinase 1 (CDK1). This review summarizes the recent findings regarding the role of integrin-mediated cell adhesion and its downstream signaling components in regulating the cell cycle, emphasizing the cell cycle progression through the G2 and early M phases. Further investigations are required to raise our knowledge about the molecular mechanisms of crosstalk between cell adhesion and the cell cycle in detail.
    Keywords:  G2 phase; adhesion; cell cycle; integrin; mitosis
    DOI:  https://doi.org/10.3390/cells11162521
  7. Front Oncol. 2022 ;12 901132
    Polo-like kinase-1, Aurora kinase A and WEE1 kinase are promising druggable targets in CML cells displaying BCR::ABL1-independent resistance to tyrosine kinase inhibitors.
    Manuela Mancini, Sara De Santis, Cecilia Monaldi, Fausto Castagnetti, Annalisa Lonetti, Samantha Bruno, Elisa Dan, Barbara Sinigaglia, Gianantonio Rosti, Michele Cavo, Gabriele Gugliotta, Simona Soverini.
      In chronic myeloid leukemia (CML), Aurora kinase A and Polo like kinase 1 (PLK1), two serine-threonine kinases involved in the maintenance of genomic stability by preserving a functional G2/M checkpoint, have been implicated in BCR::ABL1-independent resistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate and in leukemic stem cell (LSC) persistence. It can be speculated that the observed deregulated activity of Aurora A and Plk1 enhances DNA damage, promoting the occurrence of additional genomic alterations contributing to TKI resistance and ultimately driving progression from chronic phase to blast crisis (BC). In this study, we propose a new therapeutic strategy based on the combination of Aurora kinase A or PLK1 inhibition with danusertib or volasertib, respectively, and WEE1 inhibition with AZD1775. Danusertib and volasertib used as single drugs induced apoptosis and G2/M-phase arrest, associated with accumulation of phospho-WEE1. Subsequent addition of the WEE1 inhibitor AZD1775 in combination significantly enhanced the induction of apoptotic cell death in TKI-sensitive and -resistant cell lines as compared to both danusertib and volasertib alone and to the simultaneous combination. This schedule indeed induced a significant increase of the DNA double-strand break marker γH2AX, forcing the cells through successive replication cycles ultimately resulting in apoptosis. Finally, combination of danusertib or volasertib+AZD1775 significantly reduced the clonogenic potential of CD34+ CML progenitors from BC patients. Our results may have implications for the development of innovative therapeutic approaches aimed to improve the outcomes of patients with multi-TKI-resistant or BC CML.
    Keywords:  WEE1 kinase; aurora kinase A; chronic myeloid leukemia; imatinib resistance; polo like kinase-1
    DOI:  https://doi.org/10.3389/fonc.2022.901132
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