bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2024–02–11
seven papers selected by
Valentina Piano, Uniklinik Köln
  1. Co-regulation of NDC80 complex subunits determines the fidelity of the spindle-assembly checkpoint and mitosis.
  2. Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling.
  3. Artificial tethering of constitutive centromere-associated network proteins induces CENP-A deposition without Knl2 in DT40 cells.
  4. Spastin regulates anaphase chromosome separation distance and microtubule-containing nuclear tunnels.
  5. Unraveling the mechanism of centromere inactivation in human aging.
  6. Organization of microtubule plus-end dynamics by phase separation in mitosis.
  7. Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.
  1. Mol Cancer Res. 2024 Feb 07.
    Co-regulation of NDC80 complex subunits determines the fidelity of the spindle-assembly checkpoint and mitosis.
    Se Hong Kim, Thomas T Y Lau, Man Kit Liao, Hoi Tang Ma, Randy Y C Poon.
      NDC80 complex (NDC80C) is composed of four subunits (SPC24, SPC25, NDC80, and NUF2) and is vital for kinetochore-microtubule (KT-MT) attachment during mitosis. Paradoxically, NDC80C also functions in the activation of the spindle-assembly checkpoint (SAC). This raises an interesting question regarding how mitosis is regulated when NDC80C levels are compromised. Using a degron-mediated depletion system, we found that acute silencing of SPC24 triggered a transient mitotic arrest followed by mitotic slippage. SPC24-deficient cells were unable to sustain SAC activation despite the loss of KT-MT interaction. Intriguingly, our results revealed that other subunits of the NDC80C were co-downregulated with SPC24 at a post-translational level. Silencing any individual subunit of NDC80C likewise reduced expression of the entire complex. We found that SPC24-SPC25 and NDC80-NUF2 subcomplexes could be individually stabilized using ectopically expressed subunits. Synergism of SPC24 downregulation with drugs that promote either mitotic arrest or mitotic slippage further underscored the dual roles of NDC80C in KT-MT interaction and SAC maintenance. The tight coordinated regulation of NDC80C subunits suggests that targeting individual subunit could disrupt mitotic progression and provide new avenues for therapeutic intervention. Implications: These results highlight the tight coordinated regulation of NDC80C subunits and their potential as targets for antimitotic therapies.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-23-0828
  2. Commun Biol. 2024 Feb 09. 7(1): 164
    Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling.
    Yuqing Zhang, Rose Young, Dimitriya H Garvanska, Chunlin Song, Yujing Zhai, Ying Wang, Hongfei Jiang, Jing Fang, Jakob Nilsson, Claudio Alfieri, Gang Zhang.
      Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.
    DOI:  https://doi.org/10.1038/s42003-024-05859-6
  3. J Cell Sci. 2024 Feb 01. pii: jcs261639. [Epub ahead of print]137(3):
    Artificial tethering of constitutive centromere-associated network proteins induces CENP-A deposition without Knl2 in DT40 cells.
    JingHui Cao, Tetsuya Hori, Mariko Ariyoshi, Tatsuo Fukagawa.
      The kinetochore is an essential structure for chromosome segregation. Although the kinetochore is usually formed on a centromere locus, it can be artificially formed at a non-centromere locus by protein tethering. An artificial kinetochore can be formed by tethering of CENP-C or CENP-I, members of the constitutive centromere-associated network (CCAN). However, how CENP-C or CENP-I recruit the centromere-specific histone CENP-A to form an artificial kinetochore remains unclear. In this study, we analyzed this issue using the tethering assay combined with an auxin-inducible degron (AID)-based knockout method in chicken DT40 cells. We found that tethering of CENP-C or CENP-I induced CENP-A incorporation at the non-centromeric locus in the absence of Knl2 (or MIS18BP1), a component of the Mis18 complex, and that Knl2 tethering recruited CENP-A in the absence of CENP-C. We also showed that CENP-C coimmunoprecipitated with HJURP, independently of Knl2. Considering these results, we propose that CENP-C recruits CENP-A by HJURP binding to form an artificial kinetochore. Our results suggest that CENP-C or CENP-I exert CENP-A recruitment activity, independently of Knl2, for artificial kinetochore formation in chicken DT40 cells. This gives us a new insight into mechanisms for CENP-A incorporation.
    Keywords:  CENP-A; CENP-C; Centromere; HJURP; Kinetochore; Knl2
    DOI:  https://doi.org/10.1242/jcs.261639
  4. Mol Biol Cell. 2024 Feb 09. mbcE24010031T
    Spastin regulates anaphase chromosome separation distance and microtubule-containing nuclear tunnels.
    Megan E Kelley, Lina Carlini, Nikolay Kornakov, Amol Aher, Alexey Khodjakov, Tarun M Kapoor.
      Nuclear envelope reassembly during the final stages of each mitosis depends on disassembling spindle microtubules without disrupting chromosome separation. This process involves the transient recruitment of the ESCRT-III complex and spastin, a microtubule-severing AAA (ATPases associated with diverse cellular activities) mechanoenzyme, to late-anaphase chromosomes. However, dissecting mechanisms underlying these rapid dynamics has been difficult. Here we combine the use of fast-acting chemical inhibitors and live-cell imaging and find that spindle microtubules, along with spastin activity, increase the lifetimes of spastin foci at anaphase chromosomes. Unexpectedly, spastin inhibition impedes chromosome separation, but does not alter the anaphase dynamics of CHMP4B, an ESCRT-III protein, or increase γ-H2AX foci, a DNA damage marker. We show spastin inhibition increases the frequency of lamin-lined nuclear microtunnels that can include microtubules penetrating the nucleus. Our findings suggest failure to sever spindle microtubules impedes chromosome separation, yet reforming nuclear envelopes can topologically accommodate persistent microtubules ensuring nuclear DNA is not damaged or exposed to cytoplasm.
    DOI:  https://doi.org/10.1091/mbc.E24-01-0031-T
  5. bioRxiv. 2023 Dec 30. pii: 2023.12.30.573721. [Epub ahead of print]
    Unraveling the mechanism of centromere inactivation in human aging.
    Sweta Sikder, Songjoon Baek, Truman McNeil, Yamini Dalal.
      Aging involves a range of genetic, epigenetic, and physiological alterations. A key characteristic of aged cells is the loss of global heterochromatin, accompanied by a reduction in canonical histone levels. In this study, we track the fate of centromeres during aging in human cells. Our findings reveal that the centromeric histone H3 variant CENP-A is downregulated in aged cells, in a p53-dependent manner. We observe repression of centromeric noncoding transcription through an epigenetic mechanism via recruitment of a lysine-specific demethylase 1 (LSD1/KDM1A) to centromeres. This suppression results in defective de novo CENP-A loading at aging centromeres. By dual inhibition of p53 and LSD1/KDM1A in aged cells, we mitigate the reduction in centromeric proteins and centromeric transcripts, leading to mitotic rejuvenation of these cells. These results offer insights into a novel mechanism for centromeric inactivation during aging and provide potential strategies to reactivate centromeres.
    DOI:  https://doi.org/10.1101/2023.12.30.573721
  6. J Mol Cell Biol. 2024 Feb 06. pii: mjae006. [Epub ahead of print]
    Organization of microtubule plus-end dynamics by phase separation in mitosis.
    Fengrui Yang, Mingrui Ding, Xiaoyu Song, Fang Chen, Tongtong Yang, Chunyue Wang, Chengcheng Hu, Qing Hu, Yihan Yao, Shihao Du, Phil Y Yao, Peng Xia, Gregory Adams, Chuanhai Fu, Shengqi Xiang, Dan Liu, Zhikai Wang, Kai Yuan, Xing Liu.
      In eukaryotes, microtubule polymers are essential for cellular plasticity and fate decisions. End-binding (EB) proteins serve as scaffolds for orchestrating microtubule polymer dynamics and are essential for cellular dynamics and chromosome segregation in mitosis. Here, we show that EB1 forms molecular condensates with TIP150 and MCAK through liquid-liquid phase separation to compartmentalize the kinetochore-microtubule plus-end machinery, ensuring accurate kinetochore-microtubule interactions during chromosome segregation in mitosis. Perturbation of EB1-TIP150 polymer formation by a competing peptide prevents phase separation of the EB1-mediated complex and chromosome alignment at the metaphase equator in both cultured cells and Drosophila embryos. Lys220 of EB1 is dynamically acetylated by p300/CBP-associated factor in early mitosis, and persistent acetylation at Lys220 attenuates the phase separation of the EB1-mediated complex, dissolves droplets in vitro, and harnesses accurate chromosome segregation. Our data suggest a novel framework for understanding the organization and regulation of eukaryotic spindle for accurate chromosome segregation in mitosis.
    Keywords:  EB1; acetylation; microtubule dynamics; mitosis; phase separation
    DOI:  https://doi.org/10.1093/jmcb/mjae006
  7. Sci Adv. 2024 Feb 09. 10(6): eadi7840
    Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.
    Fabian Bock, Xinyu Dong, Shensen Li, Olga M Viquez, Eric Sha, Matthew Tantengco, Elizabeth M Hennen, Erin Plosa, Alireza Ramezani, Kyle L Brown, Young Mi Whang, Andrew S Terker, Juan Pablo Arroyo, David G Harrison, Agnes Fogo, Cord H Brakebusch, Ambra Pozzi, Roy Zent.
      Prolonged obstruction of the ureter, which leads to injury of the kidney collecting ducts, results in permanent structural damage, while early reversal allows for repair. Cell structure is defined by the actin cytoskeleton, which is dynamically organized by small Rho guanosine triphosphatases (GTPases). In this study, we identified the Rho GTPase, Rac1, as a driver of postobstructive kidney collecting duct repair. After the relief of ureteric obstruction, Rac1 promoted actin cytoskeletal reconstitution, which was required to maintain normal mitotic morphology allowing for successful cell division. Mechanistically, Rac1 restricted excessive actomyosin activity that stabilized the negative mitotic entry kinase Wee1. This mechanism ensured mechanical G2-M checkpoint stability and prevented premature mitotic entry. The repair defects following injury could be rescued by direct myosin inhibition. Thus, Rac1-dependent control of the actin cytoskeleton integrates with the cell cycle to mediate kidney tubular repair by preventing dysmorphic cells from entering cell division.
    DOI:  https://doi.org/10.1126/sciadv.adi7840
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