bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2023–03–12
eleven papers selected by
Valentina Piano, Uniklinik Köln
  1. On the assembly of the mitotic spindle, bistability and hysteresis.
  2. PP6 regulation of Aurora A-TPX2 limits NDC80 phosphorylation and mitotic spindle size.
  3. Structure of the Ndc80 complex and its interactions at the yeast kinetochore-microtubule interface.
  4. CENP-I directly targets centromeric DNA to support CENP-A deposition and centromere maintenance.
  5. Separase and Roads to Disengage Sister Chromatids during Anaphase.
  6. Systematic analysis of microtubule plus-end networks defines EB-cargo complexes critical for mitosis in budding yeast.
  7. EWSR1 prevents the induction of aneuploidy through direct regulation of Aurora B.
  8. Mitotic kinase inhibitors as Therapeutic Interventions for Prostate Cancer: Evidence from In vitro Studies.
  9. Polo-like kinase 1 promotes Cdc42-induced actin polymerization for asymmetric division in oocytes.
  10. Deep learning techniques and mathematical modeling allow 3D analysis of mitotic spindle dynamics.
  11. SETD2 non genomic loss of function in advanced systemic mastocytosis is mediated by an Aurora kinase A/MDM2 axis and can be therapeutically targeted.
  1. Cell Mol Life Sci. 2023 Mar 08. 80(4): 83
    On the assembly of the mitotic spindle, bistability and hysteresis.
    Angela Flavia Serpico, Caterina Pisauro, Domenico Grieco.
      During cell division, the transition from interphase to mitosis is dictated by activation of the cyclin B-cdk1 (Cdk1) complex, master mitotic kinase. During interphase, Cdk1 accumulates in an inactive state (pre-Cdk1). When Cdk1 overcomes a certain threshold of activity upon initial activation of pre-Cdk1, then the stockpiled pre-Cdk1 is rapidly converted into overshooting active Cdk1, and mitosis is established irreversibly in a switch-like fashion. This is granted by positive Cdk1 activation loops and the concomitant inactivation of Cdk1 counteracting phosphatases, empowering Cdk1 activity and favoring the Cdk1-dependent phosphorylations that are required to establish mitosis. These circuitries prevent backtracking and ensure unidirectionality so that interphase and mitosis are considered bistable states. Mitosis also shows hysteresis, meaning that the levels of Cdk1 activity needed to establish mitosis are higher than those required to maintain it; therefore, once in mitosis cells can tolerate moderate drops in Cdk1 activity without exiting mitosis. Whether these features have other functional implications in addition to the general action of preventing backtracking is unknown. Here, we contextualize these concepts in the view of recent evidence indicating that loss of activity of small and compartmentalized amounts of Cdk1 within mitosis is necessary to assemble the mitotic spindle, the structure required to segregate replicated chromosomes. We further propose that, in addition to prevent backtracking, the stability and hysteresis properties of mitosis are also essential to move forward in mitosis by allowing cells to bear small, localized, drops in Cdk1 activity that are necessary to build the mitotic spindle.
    Keywords:  Cell cycle progression; Checkpoint; Chromosome segregation; Genome stability; Spindle assembly; i-Cdk1
    DOI:  https://doi.org/10.1007/s00018-023-04727-6
  2. J Cell Biol. 2023 May 01. pii: e202205117. [Epub ahead of print]222(5):
    PP6 regulation of Aurora A-TPX2 limits NDC80 phosphorylation and mitotic spindle size.
    Tomoaki Sobajima, Katarzyna M Kowalczyk, Stefanos Skylakakis, Daniel Hayward, Luke J Fulcher, Colette Neary, Caleb Batley, Samvid Kurlekar, Emile Roberts, Ulrike Gruneberg, Francis A Barr.
      Amplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the catalytic subunit of PP6, have amplified Aurora A activity, and as we show here, enlarged mitotic spindles which fail to hold chromosomes tightly together in anaphase, causing defective nuclear structure. Using functional genomics to shed light on the processes underpinning these changes, we discover synthetic lethality between PPP6C and the kinetochore protein NDC80. We find that NDC80 is phosphorylated on multiple N-terminal sites during spindle formation by Aurora A-TPX2, exclusively at checkpoint-silenced, microtubule-attached kinetochores. NDC80 phosphorylation persists until spindle disassembly in telophase, is increased in PPP6C knockout cells, and is Aurora B-independent. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C knockout cells. In regulating NDC80 phosphorylation by Aurora A-TPX2, PP6 plays an important role in mitotic spindle formation and size control and thus the fidelity of cell division.
    DOI:  https://doi.org/10.1083/jcb.202205117
  3. Open Biol. 2023 Mar;13(3): 220378
    Structure of the Ndc80 complex and its interactions at the yeast kinetochore-microtubule interface.
    Jacob A Zahm, Simon Jenni, Stephen C Harrison.
      The conserved Ndc80 kinetochore complex, Ndc80c, is the principal link between mitotic spindle microtubules and centromere-associated proteins. We used AlphaFold 2 (AF2) to obtain predictions of the Ndc80 'loop' structure and of the Ndc80 : Nuf2 globular head domains that interact with the Dam1 subunit of the heterodecameric DASH/Dam1 complex (Dam1c). The predictions guided design of crystallizable constructs, with structures close to the predicted ones. The Ndc80 'loop' is a stiff, α-helical 'switchback' structure; AF2 predictions and positions of preferential cleavage sites indicate that flexibility within the long Ndc80c rod occurs instead at a hinge closer to the globular head. Conserved stretches of the Dam1 C terminus bind Ndc80c such that phosphorylation of Dam1 serine residues 257, 265 and 292 by the mitotic kinase Ipl1/Aurora B can release this contact during error correction of mis-attached kinetochores. We integrate the structural results presented here into our current molecular model of the kinetochore-microtubule interface. The model illustrates how multiple interactions between Ndc80c, DASH/Dam1c and the microtubule lattice stabilize kinetochore attachments.
    Keywords:  Dam1 complex; Ndc80 complex; cell division; chromosome segregation; kinetochore; structure prediction
    DOI:  https://doi.org/10.1098/rsob.220378
  4. Proc Natl Acad Sci U S A. 2023 Mar 14. 120(11): e2219170120
    CENP-I directly targets centromeric DNA to support CENP-A deposition and centromere maintenance.
    Liqiao Hu, Congcong Zhao, Mingjie Liu, Shuaiyu Liu, Jingjing Ye, Kehui Wang, Jinyun Shi, Wei Tian, Xiaojing He.
      The enrichment of histone H3 variant CENP-A is the epigenetic mark of centromere and initiates the assembly of the kinetochore at centromere. The kinetochore is a multi-subunit complex that ensures accurate attachment of microtubule centromere and faithful segregation of sister chromatids during mitosis. As a subunit of kinetochore, CENP-I localization at centromere also depends on CENP-A. However, whether and how CENP-I regulates CENP-A deposition and centromere identity remains unclear. Here, we identified that CENP-I directly interacts with the centromeric DNA and preferentially recognizes AT-rich elements of DNA via a consecutive DNA-binding surface formed by conserved charged residues at the end of N-terminal HEAT repeats. The DNA binding-deficient mutants of CENP-I retained the interaction with CENP-H/K and CENP-M, but significantly diminished the centromeric localization of CENP-I and chromosome alignment in mitosis. Moreover, the DNA binding of CENP-I is required for the centromeric loading of newly synthesized CENP-A. CENP-I stabilizes CENP-A nucleosomes upon binding to nucleosomal DNA instead of histones. These findings unveiled the molecular mechanism of how CENP-I promotes and stabilizes CENP-A deposition and would be insightful for understanding the dynamic interplay of centromere and kinetochore during cell cycle.
    Keywords:  CENP-A deposition; CENP-I; centromere; centromeric DNA; kinetochore
    DOI:  https://doi.org/10.1073/pnas.2219170120
  5. Int J Mol Sci. 2023 Feb 27. pii: 4604. [Epub ahead of print]24(5):
    Separase and Roads to Disengage Sister Chromatids during Anaphase.
    Marketa Konecna, Soodabeh Abbasi Sani, Martin Anger.
      Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.
    Keywords:  CDK1; Cyclin B1; Mad2; Sgo2; aneuploidy; chromosome division; cohesin; securin; segregation errors; separase
    DOI:  https://doi.org/10.3390/ijms24054604
  6. Mol Biol Cell. 2023 Mar 08. mbcE23020054
    Systematic analysis of microtubule plus-end networks defines EB-cargo complexes critical for mitosis in budding yeast.
    Nikolay Kornakov, Stefan Westermann.
      Microtubules are ubiquitous cytoskeletal polymers with essential functions in chromosome segregation, intracellular transport and cellular morphogenesis. End-binding proteins (EBs) form the nodes of intricate microtubule plus-end interaction networks. Which EB binding partners are most critical for cell division, and how cells organize a microtubule cytoskeleton in the absence of an EB protein, are open questions. Here, we perform a detailed analysis of deletion and point mutants of the budding yeast EB protein Bim1. We demonstrate that Bim1 executes its key mitotic functions as part of two cargo complexes - Bim1-Kar9 in the cytoplasm and Bim1-Bik1-Cik1-Kar3 in the nucleus. The latter complex acts during initial metaphase spindle assembly and supports tension establishment and sister chromatid bi-orientation. We demonstrate that engineered plus-end targeting of Cik1-Kar3 and overexpression of the microtubule crosslinker Ase1 restore distinct aspects of the bim1Δ spindle phenotype. In addition to defining key Bim1-cargo complexes our study also characterizes redundant mechanisms that allow cells to proliferate in the absence of Bim1. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E23-02-0054
  7. Front Cell Dev Biol. 2023 ;11 987153
    EWSR1 prevents the induction of aneuploidy through direct regulation of Aurora B.
    Haeyoung Kim, Hyewon Park, Evan T Schulz, Yoshiaki Azuma, Mizuki Azuma.
      EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using an Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5'-end using a CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at Kinetochore proximal centromere compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B.
    Keywords:  Aurora B; aneuploidy; auxin inducible degron (AID) system; chromosome mis-segregation; mitosis; sarcoma
    DOI:  https://doi.org/10.3389/fcell.2023.987153
  8. Endocr Metab Immune Disord Drug Targets. 2023 Mar 03.
    Mitotic kinase inhibitors as Therapeutic Interventions for Prostate Cancer: Evidence from In vitro Studies.
    Aadil Javed, Gülseren Özduman, Sevda Altun, Doğan Duran, Dilan Yerli, Tilbe Özar, Faruk Şimşek, Kemal Sami Korkmaz.
      Prostate cancer is one of the devastating diseases characterized by genetic changes leading to uncontrolled growth and metastasis of the cells of the prostate gland and affects men worldwide. Conventional hormonal and chemotherapeutic agents are effective in mitigating the disease if diagnosed at an early stage. All dividing eukaryotic cells require mitotic progression for the maintenance of genomic integrity in progeny populations. The protein kinases, upon activation and de-activation in an ordered fashion, lead to spatial and temporal regulation of the cell division process. The entry into mitosis along with the progression into sub-phases of mitosis is ensured due to the activity of mitotic kinases. These kinases include Polo-Like-Kinase 1 (PLK1), Aurora kinases, and Cyclin-Dependent-Kinase 1 (CDK1), among others. The mitotic kinases, among others, are usually overexpressed in many cancers and can be targeted using small molecule inhibitors to reduce the effects of these regulators on mechanisms, such as regulation of genomic integrity and mitotic fidelity. In this review, we attempted to discuss the appropriate functions of mitotic kinases revealed through cell culture studies and the impact of their respective inhibitors derived in pre-clinical studies. The review is designed to elucidate the growing field of small molecule inhibitors and their functional screening or mode of action at the cellular and molecular level in the context of Prostate Cancer. Therefore, studies performed specifically on cells of Prostatic-origin are narrated in this review, culminating in a comprehensive view of the specific field of mitotic kinases that can be targeted for therapy of Prostate cancer.
    Keywords:  Cancer; Cell Cycle; Cell lineProstate; Cell lines; Inhibitors; Kinases; Mitosis; Prostate
    DOI:  https://doi.org/10.2174/1871530323666230303092243
  9. Open Biol. 2023 Mar;13(3): 220326
    Polo-like kinase 1 promotes Cdc42-induced actin polymerization for asymmetric division in oocytes.
    Wai Shan Yuen, Qing Hua Zhang, Anne Bourdais, Deepak Adhikari, Guillaume Halet, John Carroll.
      Polo-like kinase I (Plk1) is a highly conserved seronine/threonine kinase essential in meiosis and mitosis for spindle formation and cytokinesis. Here, through temporal application of Plk1 inhibitors, we identify a new role for Plk1 in the establishment of cortical polarity essential for highly asymmetric cell divisions of oocyte meiosis. Application of Plk1 inhibitors in late metaphase I abolishes pPlk1 from spindle poles and prevents the induction of actin polymerization at the cortex through inhibition of local recruitment of Cdc42 and Neuronal Wiskott-Aldrich Syndrome protein (N-WASP). By contrast, an already established polar actin cortex is insensitive to Plk1 inhibitors, but if the polar cortex is first depolymerized, Plk1 inhibitors completely prevent its restoration. Thus, Plk1 is essential for establishment but not maintenance of cortical actin polarity. These findings indicate that Plk1 regulates recruitment of Cdc42 and N-Wasp to coordinate cortical polarity and asymmetric cell division.
    Keywords:  Cdc42; Plk1; actin; meiosis; oocyte; polarity
    DOI:  https://doi.org/10.1098/rsob.220326
  10. J Cell Biol. 2023 May 01. pii: e202111094. [Epub ahead of print]222(5):
    Deep learning techniques and mathematical modeling allow 3D analysis of mitotic spindle dynamics.
    David Dang, Christoforos Efstathiou, Dijue Sun, Haoran Yue, Nishanth R Sastry, Viji M Draviam.
      Time-lapse microscopy movies have transformed the study of subcellular dynamics. However, manual analysis of movies can introduce bias and variability, obscuring important insights. While automation can overcome such limitations, spatial and temporal discontinuities in time-lapse movies render methods such as 3D object segmentation and tracking difficult. Here, we present SpinX, a framework for reconstructing gaps between successive image frames by combining deep learning and mathematical object modeling. By incorporating expert feedback through selective annotations, SpinX identifies subcellular structures, despite confounding neighbor-cell information, non-uniform illumination, and variable fluorophore marker intensities. The automation and continuity introduced here allows the precise 3D tracking and analysis of spindle movements with respect to the cell cortex for the first time. We demonstrate the utility of SpinX using distinct spindle markers, cell lines, microscopes, and drug treatments. In summary, SpinX provides an exciting opportunity to study spindle dynamics in a sophisticated way, creating a framework for step changes in studies using time-lapse microscopy.
    DOI:  https://doi.org/10.1083/jcb.202111094
  11. Biomark Res. 2023 Mar 10. 11(1): 29
    SETD2 non genomic loss of function in advanced systemic mastocytosis is mediated by an Aurora kinase A/MDM2 axis and can be therapeutically targeted.
    Manuela Mancini, Cecilia Monaldi, Sara De Santis, Cristina Papayannidis, Michela Rondoni, Chiara Sartor, Samantha Bruno, Livio Pagano, Marianna Criscuolo, Roberta Zanotti, Massimiliano Bonifacio, Patrizia Tosi, Michel Arock, Peter Valent, Michele Cavo, Simona Soverini.
       BACKGROUND: The SETD2 tumor suppressor gene encodes a histone methyltransferase that safeguards transcription fidelity and genomic integrity via trimethylation of histone H3 lysine 36 (H3K36Me3). SETD2 loss of function has been observed in solid and hematologic malignancies. We have recently reported that most patients with advanced systemic mastocytosis (AdvSM) and some with indolent or smoldering SM display H3K36Me3 deficiency as a result of a reversible loss of SETD2 due to reduced protein stability.
    METHODS: Experiments were conducted in SETD2-proficient (ROSAKIT D816V) and -deficient (HMC-1.2) cell lines and in primary cells from patients with various SM subtypes. A short interfering RNA approach was used to silence SETD2 (in ROSAKIT D816V cells), MDM2 and AURKA (in HMC-1.2 cells). Protein expression and post-translational modifications were assessed by WB and immunoblotting. Protein interactions were tested by using co-immunoprecipitation. Apoptotic cell death was evaluated by flow cytometry after annexin V and propidium iodide staining, respectively. Drug cytotoxicity in in vitro experiments was evaluated by clonogenic assays.
    RESULTS: Here, we show that the proteasome inhibitors suppress cell growth and induce apoptosis in neoplastic mast cells by promoting SETD2/H3K36Me3 re-expression. Moreover, we found that Aurora kinase A and MDM2 are implicated in SETD2 loss of function in AdvSM. In line with this observation, direct or indirect targeting of Aurora kinase A with alisertib or volasertib induced reduction of clonogenic potential and apoptosis in human mast cell lines and primary neoplastic cells from patients with AdvSM. Efficacy of Aurora A or proteasome inhibitors was comparable to that of the KIT inhibitor avapritinib. Moreover, combination of alisertib (Aurora A inhibitor) or bortezomib (proteasome inhibitor) with avapritinib allowed to use lower doses of each drug to achieve comparable cytotoxic effects.
    CONCLUSIONS: Our mechanistic insights into SETD2 non-genomic loss of function in AdvSM highlight the potential value of novel therapeutic targets and agents for the treatment of patients who fail or do not tolerate midostaurin or avapritinib.
    Keywords:  Aurora kinase A; MDM2; Polo-like kinase 1; Proteasome inhibitors; SETD2
    DOI:  https://doi.org/10.1186/s40364-023-00468-7
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