bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2025–05–11
ten papers selected by
Valentina Piano, Uniklinik Köln
  1. AURKA controls oocyte spindle assembly checkpoint and chromosome alignment by HEC1 phosphorylation.
  2. SART1 uniquely localizes to spindle poles forming a SART1 cap and promotes spindle pole assembly.
  3. Engineering Chromosome Bridges Through CRISPR/Cas9 to Decipher the Impact of Intercentromeric Distance on Resolution Dynamics.
  4. The NLS3 Motif in TPX2 Regulates Spindle Architecture in Xenopus Egg Extracts.
  5. Bridging-mediated compaction of mitotic chromosomes.
  6. [m6A modification regulates PLK1 expression and mitosis].
  7. Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing.
  8. The Kinesin-14 Tail: Dual microtubule binding domains drive spindle morphogenesis through tight microtubule cross-linking and robust sliding.
  9. Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts.
  10. Cell division: Size-scaling cytoplasmic flows transport chromosomes to the right spot.
  1. Life Sci Alliance. 2025 Jul;pii: e202403146. [Epub ahead of print]8(7):
    AURKA controls oocyte spindle assembly checkpoint and chromosome alignment by HEC1 phosphorylation.
    Cecilia S Blengini, Shuang Tang, Robert J Mendola, G John Garrisi, Jason E Swain, Karen Schindler.
      In human oocytes, meiosis I is error-prone, causing early miscarriages and developmental disorders. The Aurora protein kinases are key regulators of chromosome segregation in mitosis and meiosis, and their dysfunction is associated with aneuploidy. Oocytes express three Aurora kinase (AURK) proteins, but only AURKA is necessary and sufficient to support oocyte meiosis in mice. However, the unique molecular contributions to ensuring high egg quality of AURKA remain unclear. Here, using a combination of genetic and pharmacological approaches, we evaluated how AURKA phosphorylation regulates outer kinetochore function during oocyte meiosis. We found that the outer kinetochore protein Ndc80/HEC1 is constitutively phosphorylated at multiple residues by Aurora kinases during meiosis I, but that serine 69 is specifically phosphorylated by AURKA in mouse and human oocytes. We further show that serine 69 phosphorylation contributes to spindle assembly checkpoint activation and chromosome alignment during meiosis I. These results provide a fundamental mechanistic understanding of how AURKA regulates meiosis and kinetochore function to ensure meiosis I fidelity.
    DOI:  https://doi.org/10.26508/lsa.202403146
  2. J Biol Chem. 2025 May 02. pii: S0021-9258(25)00410-7. [Epub ahead of print] 108561
    SART1 uniquely localizes to spindle poles forming a SART1 cap and promotes spindle pole assembly.
    Hideki Yokoyama, Daniel Moreno-Andrés, Kaoru Takizawa, Zhenzhen Chu, Anja Scheufen, Tsumugi Funabashi, Jian Ma, Wolfram Antonin, Oliver J Gruss, Yoshikazu Haramoto.
      The nuclear protein SART1 has been associated with pre-mRNA splicing, but SART1 RNAi knockdown results also in defects in mitotic progression, centrosome biogenesis, and chromosome cohesion. The mitotic roles of SART1 have not been characterized in detail, and it remains unclear whether SART1 functions in mitosis directly or indirectly via pre-mRNA splicing. Here, we identify SART1 as a direct, mitosis-specific microtubule-associated protein. SART1 downregulation in human cells leads to spindle assembly defects with reduced microtubule dynamics, end-on attachment defects, and checkpoint activation, while microtubule dynamics remain unaffected in interphase. SART1 uniquely localizes to the distal surface of mitotic centrosomes along the spindle axis, forming a previously not described structure we refer to as SART1 cap. Immunoprecipitation of SART1 consistently identifies centrosomal proteins as interaction partners. Immunostaining of these shows that SART1 downregulation does not affect centriole duplication and centrosome-accumulation of γ-tubulin but reduces the accumulation of selective pericentriolar material (PCM) proteins such as Ninein. Depletion of SART1 from frog egg extracts disrupts spindle pole assembly around sperm nuclei and DNA-coated beads. Spindles formed around DNA-coated beads do not contain centrosomes but still recruit PCM proteins for spindle pole assembly. We finally show that the N-terminus of SART1 is its microtubule-binding region and is essential for spindle assembly. Our data unravel a unique localization of SART1 and its novel function to recruit selective PCM proteins for spindle pole assembly in centrosomal and acentrosomal spindle assembly.
    Keywords:  PCM proteins; Ran; centrosomal protein; microtubule binding; spindle pole formation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108561
  3. FASEB J. 2025 May 15. 39(9): e70599
    Engineering Chromosome Bridges Through CRISPR/Cas9 to Decipher the Impact of Intercentromeric Distance on Resolution Dynamics.
    Teresa Anglada, Marina Rodriguez-Muñoz, Núria Pulido-Artola, Anna Genescà.
      Resolution of chromosome bridges during mitosis is a critical yet incompletely understood process with implications for genomic stability and cancer development. In this study, we investigated the impact of the bridging chromatin length on the timing and mechanism of chromosome bridge resolution. Using CRISPR/Cas9 technology, we engineered chromosome bridges with precisely defined intercentromeric distances in human RPE-1 cells. Our study revealed a decline in the frequency of chromosome bridges as cells progressed from early anaphase to late telophase, indicating resolution during mitosis. Moreover, the longer the bridging chromatin length, the higher the frequency of chromosome bridges observed at the mitotic exit, demonstrating that the size of the bridge influences its resolution during mitosis. Additionally, the separation between the bridge kinetochores needed for bridge breakage was strongly dependent on the megabase length of the bridging chromatin, with longer chromosome bridges requiring greater separation for their resolution. Given that chromosome bridge resolution occurs in a concerted manner with spindle elongation and is influenced by the length of the bridging chromatin, we posit that the traction forces generated by microtubules attaching to dicentric chromosomes play a significant role in resolving chromosome bridges during mitosis. Our study underscores the intricate interplay between chromosome bridge geometry and mechanical forces in mitotic chromosome bridge resolution. Our model offers a valuable framework for future investigations into the molecular mechanisms underlying chromosome bridge resolution, with potential implications for cancer biology and genomic stability maintenance.
    Keywords:  CRISPR/Cas9; bridging chromatin length; chromosome bridge; mitosis
    DOI:  https://doi.org/10.1096/fj.202402258RR
  4. Cytoskeleton (Hoboken). 2025 May 06.
    The NLS3 Motif in TPX2 Regulates Spindle Architecture in Xenopus Egg Extracts.
    Guadalupe E Pena, Xiao Zhou, Lauren Slevin, Christopher Brownlee, Rebecca Heald.
      A bipolar spindle composed of microtubules and many associated proteins functions to segregate chromosomes during cell division in all eukaryotes, yet both spindle size and architecture vary dramatically across different species and cell types. Targeting protein for Xklp2 (TPX2) is one candidate factor for modulating spindle microtubule organization through its roles in branching microtubule nucleation, activation of the mitotic kinase Aurora A, and association with the kinesin-5 (Eg5) motor. Here we characterize a conserved nuclear localization sequence (NLS) motif, 123KKLK126 in Xenopus laevis TPX2, which regulates astral microtubule formation and spindle pole morphology in Xenopus egg extracts. Addition of recombinant TPX2 with this sequence mutated to AALA stimulated spontaneous formation of microtubule asters and increased recruitment of phosphorylated Aurora A, pericentrin, and Eg5 to meiotic spindle poles while still binding to the regulatory transport factor importin α. We propose that TPX2 is a linchpin spindle assembly factor whose regulation contributes to the activation of multiple microtubule polymerizing and organizing proteins, generating distinct spindle architectures.
    Keywords:   TPX2 ; cell division; microtubule; spindle
    DOI:  https://doi.org/10.1002/cm.22034
  5. Nucleus. 2025 Dec;16(1): 2497765
    Bridging-mediated compaction of mitotic chromosomes.
    Giada Forte, Lora Boteva, Nick Gilbert, Peter R Cook, Davide Marenduzzo.
      Within living cells, chromosome shapes undergo a striking morphological transition, from loose and uncondensed fibers during interphase to compacted and cylindrical structures during mitosis. ATP driven loop extrusion performed by a specialized protein complex, condensin, has recently emerged as a key driver of this transition. However, while this mechanism can successfully recapitulate the compaction of chromatids during the early stages of mitosis, it cannot capture structures observed after prophase. Here we hypothesize that a condensin bridging activity plays an additional important role, and review evidence - obtained largely through molecular dynamics simulations - that, in combination with loop extrusion, it can generate compact metaphase cylinders. Additionally, the resulting model qualitatively explains the unusual elastic properties of mitotic chromosomes observed in micromanipulation experiments and provides insights into the role of condensins in the formation of abnormal chromosome structures associated with common fragile sites.
    Keywords:  Bridging activity; chromatin; common fragile sites; condensin; mitotic compaction
    DOI:  https://doi.org/10.1080/19491034.2025.2497765
  6. Sheng Wu Gong Cheng Xue Bao. 2025 Apr 25. 41(4): 1559-1572
    [m6A modification regulates PLK1 expression and mitosis].
    Xiaoli Chang, Xin Yan, Zhenyu Yang, Shuwen Cheng, Xiaofeng Zhu, Zhantong Tang, Wenxia Tian, Yujun Zhao, Yongbo Pan, Shan Gao.
      N6-methyladenosine (m6A) modification plays a critical role in cell cycle regulation, while the mechanism of m6A in regulating mitosis remains underexplored. Here, we found that the total m6A modification level in cells increased during mitosis by the liquid chromatography-mass spectrometry/mass spectrometry and m6A dot blot assays. Silencing methyltransferase-like 3 (METTL3) or METTL14 results in delayed mitosis, abnormal spindle assembly, and chromosome segregation defects by the immunofluorescence. By analyzing transcriptome-wide m6A targets in HeLa cells, we identified polo-like kinase 1 (PLK1) as a key gene modified by m6A in regulating mitosis. Specifically, through immunoblotting and RNA pulldown, m6A modification inhibits PLK1 translation via YTH N6-methyladenosine RNA binding protein 1, thus mediating cell cycle homeostasis. Demethylation of PLK1 mRNA leads to significant mitotic abnormalities. These findings highlight the critical role of m6A in regulating mitosis and the potential of m6A as a therapeutic target in proliferative diseases such as cancer.
    Keywords:  cell cycle; methyltransferase-like 3 (METTL3); mitosis; polo-like kinase 1 (PLK1)
    DOI:  https://doi.org/10.13345/j.cjb.240775
  7. Nat Commun. 2025 May 08. 16(1): 4281
    Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing.
    Henry Y Ng, Devon H Whelpley, Armin N Adly, Robert A Maxwell, David O Morgan.
      Cell cycle progression is governed by complexes of the cyclin-dependent kinases (CDKs) and their regulatory subunits cyclin and Cks1. CDKs phosphorylate hundreds of substrates, often at multiple sites. Multisite phosphorylation depends on Cks1, which binds initial priming phosphorylation sites to promote secondary phosphorylation at other sites. Here, we describe a similar role for a recently discovered phosphate-binding pocket (PP) on B-type cyclins. Mutation of the PP in Clb2, the major mitotic cyclin of budding yeast, alters bud morphology and delays the onset of anaphase. Mutation of the PP reduces multi-site phosphorylation of CDK substrates in vitro, including the Cdc16 and Cdc27 subunits of the anaphase-promoting complex/cyclosome and the Bud6 and Spa2 subunits of the polarisome. We conclude that the cyclin PP, like Cks1, controls the pattern of multisite phosphorylation on CDK substrates, thereby helping to establish the robust timing of cell-cycle events.
    DOI:  https://doi.org/10.1038/s41467-025-59700-7
  8. Mol Biol Cell. 2025 May 06. mbcE25020083
    The Kinesin-14 Tail: Dual microtubule binding domains drive spindle morphogenesis through tight microtubule cross-linking and robust sliding.
    Stephanie C Ems-McClung, MacKenzie Cassity, Anjaly Prasannajith, Claire E Walczak.
      Proper spindle assembly requires the Kinesin-14 family of motors to organize microtubules (MTs) into the bipolar spindle by cross-linking and sliding anti-parallel and parallel MTs through their motor and tail domains. How they mediate these different activities is unclear. We identified two MT binding domains (MBD1 and MBD2) within the Xenopus Kinesin-14 XCTK2 tail and found that MBD1 MT affinity was weaker than MBD2. Comparable to full-length GFP-XCTK2 wild-type protein (GX-WT), GFP-XCTK2 containing the MBD1 mutations (GX-MBD1mut) stimulated spindle assembly, localized moderately on the spindle, and formed narrow spindles. In contrast, GX-MBD2mut only partially stimulated spindle assembly, localized weakly on the spindle, and formed shorter spindles. Biochemical reconstitution of MT cross-linking and sliding demonstrated that GX-MBD2mut slid anti-parallel MTs faster than GX-WT and GX-MBD1mut. However, GX-WT and GX-MBD1mut statically cross-linked the majority of parallel MTs, whereas GX-MBD2mut equally slid and statically cross-linked parallel MTs without affecting their sliding velocity. These results provide a mechanism by which the two different MT binding domains in the Kinesin-14 tail balance anti-parallel MT sliding velocity (MBD1) and tight parallel MT cross-linking (MBD2), which are important for spindle assembly and localization, and provide a basis for characterizing how molecular motors organize MTs within the spindle. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E25-02-0083
  9. Mol Biol Cell. 2025 May 06. mbcE24090421
    Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts.
    Gabriel Guilloux, Maiko Kitaoka, Karel Mocaer, Claire Heichette, Laurence Duchesne, Rebecca Heald, Thierry Pecot, Romain Gibeaux.
      The spindle is key to cell division, ensuring accurate chromosome segregation. While its assembly and function are well studied, the mechanisms regulating spindle architecture remain elusive. Here, we investigate spindle organization differences between Xenopus laevis and tropicalis, leveraging expansion microscopy (ExM) to overcome conventional imaging limitations. We optimized an ExM protocol tailored for Xenopus egg extract spindles, refining fixation, denaturation, and gelation to achieve higher resolution while preserving spindle integrity. Our protocol enables pre-expansion immunofluorescence and is seamlessly compatible with both species. To quantitatively compare microtubule organization in expanded spindles between the two species, we developed an analysis pipeline able to characterize microtubule bundles throughout spindles. We show that X. laevis spindles exhibit overall a broader range of bundle sizes, while X. tropicalis spindles contain mostly smaller bundles. While both species show larger bundles near the spindle center, X. tropicalis spindles otherwise consist of very small bundles, whereas X. laevis spindles contain more medium-sized bundles. Altogether, our work reveals species-specific spindle architectures and suggests their adaptation to the different spindle size and chromatin amount. By enhancing resolution and minimizing artifacts, our ExM approach provides new insights into spindle morphology and a robust tool for further studying these large cellular assemblies. [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E24-09-0421
  10. Curr Biol. 2025 May 05. pii: S0960-9822(25)00357-4. [Epub ahead of print]35(9): R336-R339
    Cell division: Size-scaling cytoplasmic flows transport chromosomes to the right spot.
    Maria Isabel Arjona, Nicolas Minc.
      Early embryos undergo rounds of division, producing cells of reducing sizes that must scale the distance chromosomes segregate. A new study shows that this scaling results from a cell-size-dependent dampening of cytoplasmic flows that advect chromosomes.
    DOI:  https://doi.org/10.1016/j.cub.2025.03.041
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