bims-micesi 2025-01-26 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2025–01–26
twelve papers selected by
Valentina Piano, Uniklinik Köln

High-resolution analysis of human centromeric chromatin.
High CDC20 levels increase sensitivity of cancer cells to MPS1 inhibitors.
19S proteasome loss causes monopolar spindles through ubiquitin-independent KIF11 degradation.
Positively charged specificity site in cyclin B1 is essential for mitotic fidelity.
Insights into the role of phosphorylation on microtubule crosslinking by PRC1.
Multi-SpinX: An advanced framework for automated tracking of mitotic spindles and kinetochores in multicellular environments.
Involvement of RBP-J interacting and tubulin-associated protein in the distribution of protein regulator of cytokinesis 1 in mitotic spindles.
Sod1 deficiency in mouse oocytes during in vitro maturation increases chromosome segregation errors with a reduced BUBR1 at kinetochore.
Targeting the mitotic kinase NEK2 enhances CDK4/6 inhibitor efficacy by potentiating genome instability.
Human REXO4 is Required for Cell Cycle Progression.
Revisiting phosphoregulation of Cdc25C during M-phase induction.
Aurora B controls microtubule stability to regulate abscission dynamics in stem cells.

Life Sci Alliance. 2025 Apr;pii: e202402819. [Epub ahead of print]8(4):

High-resolution analysis of human centromeric chromatin.

Daniël P Melters, Minh Bui, Tatini Rakshit, Sergei A Grigoryev, David Sturgill, Yamini Dalal.

Centromeres are marked by the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle, the constitutive centromere-associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome conformations in vivo is unknown. Here, we purify endogenous centromeric chromatin associated with the CENP-C complex across the cell cycle and analyze the structures by single-molecule imaging and biochemical assays. CENP-C complex-bound chromatin was refractory to MNase digestion. The CENP-C complex increased in height throughout the cell cycle culminating in mitosis, and the smaller CENP-C complex corresponds to the dimensions of in vitro reconstituted constitutive centromere-associated network. In addition, we found two distinct CENP-A nucleosomal configurations; the taller variant was associated with the CENP-C complex. Finally, CENP-A mutants partially corrected CENP-C overexpression-induced centromeric transcription and mitotic defects. In all, our data support a working model in which CENP-C is critical in regulating centromere homeostasis by supporting a unique higher order structure of centromeric chromatin and altering the accessibility of the centromeric chromatin fiber for transcriptional machinery.

DOI: https://doi.org/10.26508/lsa.202402819
EMBO Rep. 2025 Jan 21.

High CDC20 levels increase sensitivity of cancer cells to MPS1 inhibitors.

Siqi Zheng, Linoy Raz, Lin Zhou, Yael Cohen-Sharir, Ruifang Tian, Marica Rosaria Ippolito, Sara Gianotti, Ron Saad, Rene Wardenaar, Mathilde Broekhuis, Maria Suarez Peredo Rodriguez, Soraya Wobben, Anouk van den Brink, Petra Bakker, Stefano Santaguida, Floris Foijer, Uri Ben-David.

  Spindle assembly checkpoint (SAC) inhibitors are a recently developed class of drugs, which perturb chromosome segregation during cell division, induce chromosomal instability (CIN), and eventually lead to cell death. The molecular features that determine cellular sensitivity to these drugs are not fully understood. We recently reported that aneuploid cancer cells are preferentially sensitive to SAC inhibition. Here we report that sensitivity to SAC inhibition by MPS1 inhibitors is largely driven by the expression of CDC20, a main mitotic activator of the anaphase-promoting complex (APC/C), and that the effect of CDC20 is larger than that of the APC/C itself. Mechanistically, we discovered that CDC20 depletion prolongs metaphase duration, diminishes mitotic errors, and reduces sensitivity to SAC inhibition. We found that aneuploid cells express higher basal levels of CDC20, which shortens the duration of metaphase and leads to multiple mitotic errors, resulting in increased long-term sensitivity to the additional CIN induced by SAC inhibition. Our findings propose high CDC20 expression as a molecular feature associated with the sensitivity to SAC inhibition therapy and as a potential aneuploidy-induced cellular vulnerability.

Keywords:  Aneuploidy; Cancer; Cell Cycle; Chromosomal Instability; Spindle Assembly Checkpoint (SAC)

DOI:  https://doi.org/10.1038/s44319-024-00363-8
bioRxiv. 2025 Jan 09. pii: 2025.01.08.632038. [Epub ahead of print]

19S proteasome loss causes monopolar spindles through ubiquitin-independent KIF11 degradation.

Océane Marescal, Iain M Cheeseman.

To direct regulated protein degradation, the 26S proteasome recognizes ubiquitinated substrates through its 19S particle and then degrades them in the 20S enzymatic core. Despite this close interdependency between proteasome subunits, we demonstrate that knockouts from different proteasome subcomplexes result in distinct highly cellular phenotypes. In particular, depletion of 19S PSMD lid proteins, but not that of other proteasome subunits, prevents bipolar spindle assembly during mitosis, resulting in a mitotic arrest. We find that the monopolar spindle phenotype is caused by ubiquitin-independent proteasomal degradation of the motor protein KIF11 upon loss of 19S proteins. Thus, negative regulation of 20S-mediated proteasome degradation is essential for mitotic progression and 19S and 20S proteasome components can function independently outside of the canonical 26S structure. This work reveals a role for the proteasome in spindle formation and identifies the effects of ubiquitin-independent degradation on cell cycle control.

DOI: https://doi.org/10.1101/2025.01.08.632038
Nat Commun. 2025 Jan 20. 16(1): 853

Positively charged specificity site in cyclin B1 is essential for mitotic fidelity.

Christian Heinzle, Anna Höfler, Jun Yu, Peter Heid, Nora Kremer, Rebecca Schunk, Florian Stengel, Tanja Bange, Andreas Boland, Thomas U Mayer.

Phosphorylation of substrates by cyclin-dependent kinases (CDKs) is the driving force of cell cycle progression. Several CDK-activating cyclins are involved, yet how they contribute to substrate specificity is still poorly understood. Here, we discover that a positively charged pocket in cyclin B1, which is exclusively conserved within B-type cyclins and binds phosphorylated serine- or threonine-residues, is essential for correct execution of mitosis. HeLa cells expressing pocket mutant cyclin B1 are strongly delayed in anaphase onset due to multiple defects in mitotic spindle function and timely activation of the E3 ligase APC/C. Pocket integrity is essential for APC/C phosphorylation particularly at non-consensus CDK1 sites and full in vitro ubiquitylation activity. Our results support a model in which cyclin B1's pocket facilitates sequential substrate phosphorylations involving initial priming events that assist subsequent pocket-dependent phosphorylations even at non-consensus CDK1 motifs.

DOI: https://doi.org/10.1038/s41467-024-55669-x
Mol Biol Cell. 2025 Jan 22. mbcE24120565

Insights into the role of phosphorylation on microtubule crosslinking by PRC1.

Ellinor Tai, Austin Henglein, Angus Alfieri, Gauri Saxena, Scott Forth.

The mitotic spindle is composed of distinct networks of microtubules, including interpolar bundles that can bridge sister kinetochore fibers and bundles that organize the spindle midzone in anaphase. The crosslinking protein PRC1 can mediate such bundling interactions between antiparallel microtubules. PRC1 is a substrate of mitotic kinases including CDK/cyclin-B, suggesting that it can be phosphorylated in metaphase and dephosphorylated in anaphase. How these biochemical changes to specific residues regulate its function and ability to organize bundles has been unclear. Here, we perform biophysical analyses on microtubule networks crosslinked by two PRC1 constructs, one a wild-type reflecting a dephosphorylated state, and one phosphomimetic construct with two threonine to glutamic acid substitutions near PRC1's microtubule binding domain. We find that the wild-type construct builds longer and larger bundles that form more rapidly and are much more resistant to mechanical disruption than the phosphomimetic PRC1. Interestingly, microtubule pairs organized by both constructs behave similarly within the same assays. Our results suggest that phosphorylation of PRC1 in metaphase could tune the protein to stabilize smaller and more flexible bundles, while removal of these PTMs in anaphase would promote the assembly of larger, more mechanically robust bundles to resist chromosome and pole separation forces at the spindle midzone.

DOI: https://doi.org/10.1091/mbc.E24-12-0565
Comput Biol Med. 2025 Jan 22. pii: S0010-4825(24)01711-6. [Epub ahead of print]186 109626

Multi-SpinX: An advanced framework for automated tracking of mitotic spindles and kinetochores in multicellular environments.

Binghao Chai, Christoforos Efstathiou, Muntaqa S Choudhury, Kinue Kuniyasu, Saakshi Sanjay Jain, Alexia-Cristina Maharea, Kozo Tanaka, Viji M Draviam.

  SpinX, an AI-guided spindle tracking software, allows the 3-dimensional (3D) tracking of metaphase spindle movements in mammalian cells. Using over 900 images of dividing cells, we create the Multi-SpinX framework to significantly expand SpinX's applications: a) to track spindles and cell cortex in multicellular environments, b) to combine two object tracking (spindle with kinetochores marked by centromeric probes) and c) to extend spindle tracking beyond metaphase to prometaphase and anaphase stages where spindle morphology is different. We have used a human-in-the-loop approach to assess our optimisation steps, to manually identify challenges and to build a robust computational pipeline for segmenting kinetochore pairs and spindles. Spindles of both H1299 and RPE1 cells have been assessed and validated for use through Multi-SpinX, and we expect the tool to be versatile in enabling quantitative studies of mitotic subcellular dynamics.

Keywords:  Cell cortex; High-throughput image analysis; Kinetochore tracker; Mitotic spindle tracker

DOI:  https://doi.org/10.1016/j.compbiomed.2024.109626
Front Cell Dev Biol. 2024 ;12 1472340

Involvement of RBP-J interacting and tubulin-associated protein in the distribution of protein regulator of cytokinesis 1 in mitotic spindles.

Julia Caspers, Andreas Ritter, Badi Bahrami, Samira Catharina Hoock, Susanne Roth, Alexandra Friemel, Franz Oswald, Frank Louwen, Nina-Naomi Kreis, Juping Yuan.

  The protein regulator of cytokinesis 1 (PRC1) is a key regulator of microtubule crosslinking and bundling, which is crucial for spindle formation and cytokinesis. RITA, the RBP-J interacting and tubulin-associated protein, is a microtubule associated protein. We have reported that RITA localizes to mitotic spindles modulating microtubule dynamics and stability as well as to spindle poles affecting the activity of Aurora A. As defective chromosome congression and segregation are the most remarkable features of cells depleted of RITA, we aimed to explore further potential related mechanisms, using various cellular and molecular techniques, including clustered regularly interspaced short palindromic repeats technique/deactivated CRISPR-associated protein 9 (CRISPR/dCas9), mass spectrometry, confocal microscopy, immunofluorescence, immunoprecipitation and Western blot analysis. Here, we show that FLAG-RITA precipitates PRC1 and tubulin, and that these two proteins co-localize in the central region of the central spindle. Reduction of RITA enlarges the staining area of PRC1 in mitotic spindles as well as in the central spindle. Its suppression reduces the inter-centromere distance in metaphase cells. Interestingly, microtubule bundles of the central spindle are often less organized in a non-parallel pattern, as evidenced by increased angles, relative to corresponding separating chromosomes. These data suggest a novel role for RITA in mitotic distribution of PRC1 and that its deregulation may contribute to defective chromosome movement during mitosis. As both RITA and PRC1 are closely associated with malignant progression, further work is required to elucidate the detailed molecular mechanisms by which RITA acts as a modulator in central spindle formation and cytokinesis.

Keywords:  PRC1; RITA; cytokinesis failure; mitotic defects; the central spindle

DOI:  https://doi.org/10.3389/fcell.2024.1472340
Reprod Med Biol. 2025 Jan-Dec;24(1):24(1): e12622

Sod1 deficiency in mouse oocytes during in vitro maturation increases chromosome segregation errors with a reduced BUBR1 at kinetochore.

Mitsuru Nago, Masumi Yanai, Mika Ishii, Yasuko Sato, Kazuharu Odajima, Naoko Kimura.

   Purpose: This study aimed to investigate the molecular mechanisms associated with chromosome segregation errors caused by intrinsic oxidative stress during in vitro oocyte maturation (IVM) using oocytes from Sod1-deficient (Sod1KO) mice.
Methods: Ovulated or in vitro matured cumulus-cells oocyte complexes (COCs) were collected from wild-type (WT) and Sod1KO mice and evaluated chromosome alignment, chromosome segregation, meiotic progression, and BUBR1 and REC8 protein expression levels.
Results: In 21% O2 IVM, the Sod1KO had significantly higher frequencies of chromosome misalignment and segregation errors compared to the WT, and they also reached Germinal Vesicle Break Down (GVBD) and M I stages peak earlier and showed a shorter M I stage residence time compared to the WT. These changes were associated with a decrease in the recruitment of BUBR1 to kinetochores at M I stage, but there were no differences in the expression of REC8 between the two genotypes. Furthermore, the addition of L-ascorbic acid (AsA) or N-acetyl-L-cysteine (NAC) during IVM reduced the frequency of chromosome segregation errors in Sod1KO oocytes.
Conclusions: Oxidative stress caused by Sod1 deficiency during IVM impairs the spindle assembly checkpoint function due to a decrease in the recruitment of BUBR1 to M I stage kinetochores, leading to abnormalities in meiotic progression and chromosome segregation.

Keywords:  BUBR1; chromosome segregation errors; oocyte; spindle assembly checkpoint; superoxide dismutase‐1

DOI:  https://doi.org/10.1002/rmb2.12622
J Biol Chem. 2025 Jan 16. pii: S0021-9258(25)00043-2. [Epub ahead of print] 108196

Targeting the mitotic kinase NEK2 enhances CDK4/6 inhibitor efficacy by potentiating genome instability.

Jessica R Bobbitt, Leslie Cuellar-Vite, Kristen L Weber-Bonk, Marlee R Yancey, Parth R Majmudar, Ruth A Keri.

  Selective inhibitors that target cyclin dependent kinases 4 and 6 (CDK4/6i) are FDA approved for treatment of a subset of breast cancers and are being evaluated in numerous clinical trials for other cancers. Despite this advance, a subset of tumors are intrinsically resistant to these drugs and acquired resistance is nearly inevitable. Recent mechanistic evidence suggests that in addition to stalling the cell cycle, the anti-tumor effects of CDK4/6i involve the induction of chromosomal instability (CIN). Here, we exploit this mechanism by combining CDK4/6i with other instability-promoting agents to induce maladaptive CIN and irreversible cell fates. Specifically, dual targeting of CDK4/6 and the mitotic kinase NEK2 in vitro drives centrosome amplification and the accumulation of CIN that induces catastrophic mitoses, cell cycle exit, and cell death. Dual targeting also induces CIN in vivo and significantly decreases mouse tumor volume to a greater extent than either drug alone, without inducing overt toxicity. Importantly, we provide evidence that breast cancer cells are selectively dependent on NEK2, but non-transformed cells are not, in contrast with other mitotic kinases that are commonly essential in all cell types. These findings implicate NEK2 as a potential therapeutic target for breast cancer that could circumvent the dose-limiting toxicities that are commonly observed when blocking other mitotic kinases. Moreover, these data suggest that NEK2 inhibitors could be used to sensitize tumors to FDA-approved CDK4/6i for the treatment of breast cancers, improving their efficacy and providing a foundation for expanding the patient population that could benefit from CDK4/6i.

Keywords:  CDK4/6 inhibitors; NEK2; breast cancer; cell cycle; cell death; combination therapy; genomic instability; mitosis; mitotic catastrophe

DOI:  https://doi.org/10.1016/j.jbc.2025.108196
bioRxiv. 2025 Jan 09. pii: 2025.01.08.631954. [Epub ahead of print]

Human REXO4 is Required for Cell Cycle Progression.

Kevin M Clutario, Mai Abdusamad, Ivan Ramirez, Kayla J Rich, Ankur A Gholkar, Julian Zaragoza, Jorge Z Torres.

Human REXO4 is a poorly characterized exonuclease that is overexpressed in human cancers. To better understand the function of REXO4 and its relationship to cellular proliferation, we have undertaken multidisciplinary approaches to characterize its cell cycle phase-dependent subcellular localization and the cis determinants required for this localization, its importance to cell cycle progression and cell viability, its protein-protein association network, and its activity. We show that the localization of REXO4 to the nucleolus in interphase depends on an N-terminal nucleolar localization sequence and that its localization to the perichromosomal layer of mitotic chromosomes is dependent on Ki67. Depletion of REXO4 led to a G1/S cell cycle arrest, and reduced cell viability. REXO4 associated with ribosome components and other proteins involved in rRNA metabolism. We propose a model where REXO4 is important for proper rRNA processing, which is required for ribosome biogenesis, cell cycle progression, and proliferation.
SIGNIFICANCE STATEMENT: REXO4 is a putative RNA exonuclease with limited characterization. The authors used in silico, cell, and molecular biology approaches to characterize its localization, associations, regulation, and function.They found that during interphase, REXO4 localizes to the nucleolus through an N-terminal nucleolar localization sequence. Whereas during mitosis, REXO4 localized to the perichromosomal layer in a Ki67-dependent manner. REXO4 was required for proper cell cycle progression, and viability.These results indicated that REXO4 is important for regulating cell proliferation.

DOI: https://doi.org/10.1101/2025.01.08.631954
iScience. 2025 Jan 17. 28(1): 111603

Revisiting phosphoregulation of Cdc25C during M-phase induction.

Tan Tan, Chuanfen Wu, Ruoning Wang, Bih-Fang Pan, David Hawke, Fumin Yin, Zehao Su, Boye Liu, Sue-Hwa Lin, Wei Zhang, Jian Kuang.

  Cdc25C undergoes a sudden and substantial gel mobility shift at M-phase onset, correlating with abrupt activation of both Cdc25C and Cdk1 activities. A positive feedback loop between Cdk1 and Cdc25C has been used to explain this hallmark phenomenon. Here, we demonstrate that the M-phase supershift and robust activation of Cdc25C are due to the site-comprehensive phosphorylation of its long intrinsically disordered regulatory domain without requiring Cdk1 or other major mitotic kinase activities. The phosphorylation process involves substrate-mediated assembly of phosphorylation machinery that catalyzes multisite phosphorylation continuously without substrate dissociation. In contrast to the site-comprehensive phosphorylation of Cdc25C occurring at M-phase onset, the site-specific phosphorylation of Cdc25C by Cdk1 or other major mitotic kinases generates slight gel mobility shifts and modest activation of Cdc25C prior to M-phase onset. These findings suggest a two-stage framework consisting of site-specific phosphorylation followed by site-comprehensive phosphorylation for Cdc25C regulation during M-phase induction.

Keywords:  Biological sciences; Cell; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2024.111603
Cell Rep. 2025 Jan 23. pii: S2211-1247(25)00009-9. [Epub ahead of print]44(2): 115238

Aurora B controls microtubule stability to regulate abscission dynamics in stem cells.

Snježana Kodba, Amber Öztop, Eri van Berkum, Eugene A Katrukha, Malina K Iwanski, Wilco Nijenhuis, Lukas C Kapitein, Agathe Chaigne.

  Abscission is the last step of cell division. It separates the two sister cells and consists of cutting the cytoplasmic bridge. Abscission is mediated by the ESCRT membrane remodeling machinery, which also triggers the severing of a thick bundle of microtubules. Here, we show that rather than being passive actors in abscission, microtubules control abscission speed. Using mouse embryonic stem cells, which transition from slow to fast abscission during exit from naive pluripotency, we investigate the molecular mechanism for the regulation of abscission dynamics and identify crosstalk between Aurora B activity and microtubule stability. We demonstrate that naive stem cells maintain high Aurora B activity on the bridge after cytokinesis. This high Aurora B activity leads to transient microtubule stabilization that delays abscission by decreasing MCAK recruitment to the midbody. In turn, stable microtubules promote the activity of Aurora B. Overall, our data demonstrate that Aurora B-dependent microtubule stability controls abscission dynamics.

Keywords:  Aurora B; CP: Cell biology; CP: Stem cell research; MCAK; abscission; cytoplasmic bridges; microtubules; stem cells

DOI:  https://doi.org/10.1016/j.celrep.2025.115238