bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2023‒12‒17
ten papers selected by
Valentina Piano, Uniklinik Köln
  1. Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules.
  2. Hypothesis: The opposing pulling forces exerted by spindle microtubules can cause sliding of chromatin layers and facilitate sister chromatid resolution.
  3. Eph signal inhibition potentiates the growth-inhibitory effects of PLK1 inhibition toward cancer cells.
  4. METTL16-mediated N6-methyladenosine modification of Soga1 enables proper chromosome segregation and chromosomal stability in colorectal cancer.
  5. CENP-A and CENP-B collaborate to create an open centromeric chromatin state.
  6. The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.
  7. Polo-like kinase 1 regulates immune synapse assembly and cytotoxic T cell function by driving microtubule dynamics.
  8. Development of MPS1 Inhibitors: Recent Advances and Perspectives.
  9. New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies.
  10. SUMOylation Is Enriched in the Nuclear Matrix and Required for Chromosome Segregation.
  1. Science. 2023 Dec 08. 382(6675): 1184-1190
    Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules.
    Kyle W Muir, Christopher Batters, Tom Dendooven, Jing Yang, Ziguo Zhang, Alister Burt, David Barford.
      Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.
    DOI:  https://doi.org/10.1126/science.adj8736
  2. Front Genet. 2023 ;14 1321260
    Hypothesis: The opposing pulling forces exerted by spindle microtubules can cause sliding of chromatin layers and facilitate sister chromatid resolution.
    Joan-Ramon Daban.
      Previous studies indicated that mitotic chromosome structure consists of many stacked layers formed by a mononucleosome sheet folded as a helicoid. This multilayer chromatin structure justifies the cylindrical shape of chromosomes and the transverse orientation of cytogenetic bands, and can explain chromosome duplication by the formation of a transient double helicoid that is split into two sister chromatids in mitosis. Here it is hypothesized that the bipolar pulling forces exerted by the mitotic spindle cause the sliding of the layers and facilitate sister chromatid resolution. This hypothesis is supported by three favorable conditions: i) There is no topological entanglement of DNA between adjacent layers; ii) The orientation (parallel to the stacked layers) of the bipolar kinetochore microtubules is adequate to produce layer sliding in opposite directions; iii) The viscous resistance to the sliding caused by the weak interactions between nucleosomes in adjacent layers can be overcome by the microtubule pulling forces.
    Keywords:  chromosome structure; mitosis; mitotic chromosome; multilayer chromatin; multilayer chromosome; sister chromatid resolution
    DOI:  https://doi.org/10.3389/fgene.2023.1321260
  3. Eur J Pharmacol. 2023 Dec 08. pii: S0014-2999(23)00743-4. [Epub ahead of print]963 176229
    Eph signal inhibition potentiates the growth-inhibitory effects of PLK1 inhibition toward cancer cells.
    Ryuzaburo Yuki, Hiroki Kuwajima, Ryoko Ota, Yuki Ikeda, Youhei Saito, Yuji Nakayama.
      Anti-mitotic drugs are clinically used as anti-cancer treatments. Polo-like kinase 1 (PLK1) is a promising target against cancer cell division due to its importance in the whole process of mitosis, and thus PLK1-targeting agents have been developed in the last few decades. Clinical trial studies show that several PLK1 inhibitors are generally well-tolerated. However, the response rates are limited; therefore, it is needed to improve the efficacy of those drugs. Here, we show that NVP-BHG712, an erythropoietin-producing human hepatocellular (Eph) signaling inhibitor, potentiates the growth-inhibitory effects of the PLK1 inhibitors BI2536 and BI6727 in cancer cells. This combination treatment strongly suppresses cancer spheroid formation. Moreover, the combination drastically arrests cells at mitosis by continuous activation of the spindle assembly checkpoint (SAC), thereby inducing apoptosis. SAC activation caused by the combination of NVP-BHG712 and BI2536 is due to the inhibition of centrosome maturation and separation. Although the inactivation level of the PLK1 kinase is comparable between BI2536 treatment alone and combination treatment, the combination treatment strongly inactivates MAPK signaling in mitosis. Since inhibition of MAPK signaling potentiates the efficacy of BI2536 treatment, inactivation of PLK1 kinase and MAPK signaling contributes to the strong inhibition of centrosome separation. These results suggest that Eph signal inhibition potentiates the effect of PLK1 inhibition, leading to strong mitotic arrest via SAC activation and the subsequent reduction of cancer cell survival. The combination of PLK1 inhibition and Eph signal inhibition will provide a new effective strategy for targeting cancer cell division.
    Keywords:  Cancer; Centrosome separation; Eph receptor; MAPK; PLK1; Spindle assembly checkpoint
    DOI:  https://doi.org/10.1016/j.ejphar.2023.176229
  4. Cell Prolif. 2023 Dec 12. e13590
    METTL16-mediated N6-methyladenosine modification of Soga1 enables proper chromosome segregation and chromosomal stability in colorectal cancer.
    Jimin Li, Fang Yang, Zeyu Wang, Siqing Zheng, Shuang Zhang, Chen Wang, Bing He, Jia-Bei Wang, Hao Wang.
      N6-methyladenosine (m6A) is the most prevalent internal modification in mammalian messenger RNAs and is associated with numerous biological processes. However, its role in chromosomal instability remains to be established. Here, we report that an RNA m6A methyltransferase, METTL16, plays an indispensable role in the progression of chromosome segregation and is required to preserve chromosome stability in colorectal cancer (CRC) cells. Depletion or inhibition of the methyltransferase activity of METTL16 results in abnormal kinetochore-microtubule attachment during mitosis, leading to delayed mitosis, lagging chromosomes, chromosome mis-segregation and chromosomal instability. Mechanistically, METTL16 exerts its oncogenic effects by enhancing the expression of suppressor of glucose by autophagy 1 (Soga1) in an m6A-dependent manner. CDK1 phosphorylates Soga1, thereby triggering its direct interaction with the polo box domain of PLK1. This interaction facilitates PLK1 activation and promotes mitotic progression. Therefore, targeting the METTL16-Soga1 pathway may provide a potential treatment strategy against CRC because of its essential role in maintaining chromosomal stability.
    DOI:  https://doi.org/10.1111/cpr.13590
  5. Nat Commun. 2023 Dec 12. 14(1): 8227
    CENP-A and CENP-B collaborate to create an open centromeric chromatin state.
    Harsh Nagpal, Ahmad Ali-Ahmad, Yasuhiro Hirano, Wei Cai, Mario Halic, Tatsuo Fukagawa, Nikolina Sekulić, Beat Fierz.
      Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.
    DOI:  https://doi.org/10.1038/s41467-023-43739-5
  6. Nucleic Acids Res. 2023 Dec 12. pii: gkad1182. [Epub ahead of print]
    The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.
    H Diego Folco, Hua Xiao, David Wheeler, Hanqiao Feng, Yawen Bai, Shiv I S Grewal.
      Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.
    DOI:  https://doi.org/10.1093/nar/gkad1182
  7. J Cell Sci. 2023 Dec 12. pii: jcs.261476. [Epub ahead of print]
    Polo-like kinase 1 regulates immune synapse assembly and cytotoxic T cell function by driving microtubule dynamics.
    Fabrizia Zevolini, Anna Onnis, Roxana Khazen, Sabina Müller, Giuseppe Marotta, Salvatore Valitutti, Francesca Finetti, Cosima T Baldari.
      Elimination of virally infected or tumoral cells is mediated by cytotoxic T cells (CTL). Upon antigen recognition CTLs assemble a specialized signaling and secretory domain at the interface with their target, the immune synapse (IS). During IS formation CTLs acquire a transient polarity, marked by re-orientation of the centrosome and microtubule cytoskeleton toward the IS, thus directing the transport and delivery of the lytic granules to the target cell. Based on the implication of the kinase Aurora-A in CTL function we hypothesized that its substrate, the mitotic regulator Polo-like kinase 1 (PLK1), may participate in CTL IS assembly. We demonstrate that PLK1 is phosphorylated upon TCR triggering and polarizes to the IS. PLK1 silencing or inhibition results in impaired IS assembly and function, as witnessed by defective synaptic accumulation of TCRs as well as compromised centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing. This function is achieved by coupling early signaling to microtubule dynamics, a function pivotal for CTL-mediated cytotoxicity. These results identify PLK1 as a new player in CTL IS assembly and function.
    Keywords:  Centrosome; Cytotoxic T cell; Immune synapse; Microtubules; PLK1
    DOI:  https://doi.org/10.1242/jcs.261476
  8. J Med Chem. 2023 Dec 14.
    Development of MPS1 Inhibitors: Recent Advances and Perspectives.
    Yangjie Zeng, Xiaodong Ren, Pengyao Jin, Yali Zhang, Ming Zhuo, Jubo Wang.
      Monopolar spindle kinase 1 (MPS1) plays a pivotal role as a dual-specificity kinase governing spindle assembly checkpoint activation and sister chromatid separation in mitosis. Its overexpression has been observed in various human malignancies. MPS1 reduces spindle assembly checkpoint sensitivity, allowing tumor cells with a high degree of aneuploidy to complete mitosis and survive. Thus, MPS1 has emerged as a promising candidate for cancer therapy. Despite the identification of numerous MPS1 inhibitors, only five have advanced to clinical trials with none securing FDA approval for cancer treatment. In this perspective, we provide a concise overview of the structural and functional characteristics of MPS1 by highlighting its relevance to cancer. Additionally, we explore the structure-activity relationships, selectivity, and pharmacokinetics of MPS1 inhibitors featuring diverse scaffolds. Moreover, we review the reported work on enhancing MPS1 inhibitor selectivity, offering valuable insights into the discovery of novel, highly potent small-molecule MPS1 inhibitors.
    DOI:  https://doi.org/10.1021/acs.jmedchem.3c00963
  9. Stem Cell Res Ther. 2023 Dec 13. 14(1): 368
    New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies.
    Marija Zekušić, Marina Bujić Mihica, Marija Skoko, Kruno Vukušić, Patrik Risteski, Jelena Martinčić, Iva M Tolić, Krešo Bendelja, Snježana Ramić, Tamara Dolenec, Ivana Vrgoč Zimić, Dominik Puljić, Ivanka Petric Vicković, Renata Iveković, Ivanka Batarilo, Uršula Prosenc Zmrzljak, Alan Hoffmeister, Tiha Vučemilo.
      BACKGROUND: Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.METHODS: After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.
    RESULTS: Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.
    CONCLUSION: This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.
    Keywords:  Advanced therapy medicinal products; Limbal graft; Limbal stem cell; Limbal stem cell deficiency; p63
    DOI:  https://doi.org/10.1186/s13287-023-03586-z
  10. J Biol Chem. 2023 Dec 08. pii: S0021-9258(23)02575-9. [Epub ahead of print] 105547
    SUMOylation Is Enriched in the Nuclear Matrix and Required for Chromosome Segregation.
    Zhaosu Chen, Jing Luo, Yunpeng Zhang, Shaoqi Zheng, Huifang Zhang, Yuanyong Huang, Jiemin Wong, Jiwen Li.
      As an important post-translational modification, SUMOylation plays critical roles in almost all biological processes. Although it has been well-documented that SUMOylated proteins are mainly localized in the nucleus and have roles in chromatin-related processes, we showed recently that the SUMOylation machinery is actually enriched in the nuclear matrix rather than chromatin. Here, we provide compelling biochemical, cellular imaging, and proteomic evidence that SUMOylated proteins are highly enriched in the nuclear matrix. We demonstrated that inactivation of SUMOylation by inhibiting SUMO-activating E1 enzyme or knockout of SUMO-conjugating E2 enzyme UBC9 have only mild effect on nuclear matrix composition, indicating that SUMOylation is neither required for nuclear matrix formation nor for targeting proteins to nuclear matrix. Further characterization of UBC9 knockout cells revealed that loss of SUMOylation did not result in significant DNA damage, but led to mitotic arrest and chromosome missegregation. Altogether, our study demonstrates that SUMOylated proteins are selectively enriched in the nuclear matrix and suggests a role of nuclear matrix in mediating SUMOylation and its regulated biological processes.
    Keywords:  SUMOylated proteins; SUMOylation; chromatin; histone deacetylation; nuclear matrix
    DOI:  https://doi.org/10.1016/j.jbc.2023.105547
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