bims-micesi 2025-12-07 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2025–12–07
ten papers selected by
Valentina Piano, Uniklinik Köln

Phosphorylation remodels the mitotic centrosome matrix to generate bipartite γ-tubulin complex docking sites.
Analyses of bent spindles reveal the mechanics of anaphase B in fission yeast.
Protocol for the generation of low-input Hi-C sequencing libraries of FACS-isolated mitotic cells.
Long-tailed class I myosins rely on tail-mediated phosphoinositide recognition for specific membrane recruitment.
O-GlcNAcylation of the tumor suppressor LATS1 drives mitotic progression via PLK1.
Quantitative and qualitative methods for measuring chromosomal instability in tumor cells.
Cell cycle timing and centromere positioning in Bos taurus zygotes derived from in vitro fertilization.
Lipid mapping of cell mitosis by non-covalent migratory fluorescence labelling.
MASTL Promotes Hepatocellular Carcinoma Progression and Paclitaxel Resistance Through Mitotic Catastrophe.
Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and cell cycle progression.

bioRxiv. 2025 Nov 21. pii: 2025.11.20.689565. [Epub ahead of print]

Phosphorylation remodels the mitotic centrosome matrix to generate bipartite γ-tubulin complex docking sites.

Midori Ohta, Orie Arakawa, Yajie Gu, Wanying Tian, Kevin D Corbett, Arshad Desai, Karen Oegema.

Mitotic centrosomes consist of centrioles surrounded by a proteinaceous matrix that docks and activates γ-tubulin complexes (γTuCs) to nucleate microtubules for spindle assembly. During mitotic entry, phosphorylation at centrosomes remodels CDK5RAP2 family matrix proteins to generate γTuC docking sites. We address the mechanism of this conversion using C. elegans SPD-5 as a model. We show that SPD-5 contains two regions, PRGB1 and PRGB2, that are each sufficient for Polo-Like Kinase 1 (PLK1) phosphorylation-regulated γTuC binding. We define key phosphosites in each region and uncover autoinhibition mediated by interactions within and between them. PRGB2 is dimeric and requires γTuCs containing the Mozart family microprotein MZT-1 for binding, whereas PRGB1 is monomeric and binds independently of MZT-1. Our results support PLK1 phosphorylation inducing a conformational change that enables MZT-1-dependent PRGB2 binding, which in turn relieves PRGB1 inhibition. Such a multi-step mechanism would ensure robust spindle assembly by restricting microtubule nucleation in space and time.

DOI: https://doi.org/10.1101/2025.11.20.689565
bioRxiv. 2025 Nov 20. pii: 2025.11.20.689516. [Epub ahead of print]

Analyses of bent spindles reveal the mechanics of anaphase B in fission yeast.

Paula Real-Calderón, Thomas G Fai, Rafael R Daga, Joël Lemière, Fred Chang.

The mitotic spindle in the fission yeast Schizosaccharomyces pombe is a single bundle of microtubules which elongates to segregate the chromosomes during anaphase B. The mechanical properties of the spindle and the forces driving its elongation remain poorly defined. Here, we analyzed how spindles react to mechanical and genetic perturbations to uncover their mechanical properties. Treatment of cells with osmotic oscillations and blue light led to a consistent phenotype of spindle buckling and breakage in mid-anaphase. The stalling of pole separation and reduced rates of spindle elongation indicated that spindles elongate and buckle under increased mechanical load. The structural integrity of the bent spindles was dependent on Ase1 (PRC1), while the spindle elongation rate was dependent on motor proteins Klp9 (kinesin-6) and Cut7 (kinesin-5). Modeling of bent spindle shapes revealed that most spindles behave mechanically as a beam with a two-fold increase in rigidity in the midzone. Upon reaching a threshold size, bent spindles broke at a specific fragile site near the edge of the spindle midzone. Our findings in this simple fission yeast spindle are relevant to the mechanics of more complex metazoan spindles.
Significance statement: The anaphase B spindle in S. pombe consists of a microtubule bundle that elongates to move the chromosomes apart. The various forces and mechanical properties of the spindle remain poorly quantified. The authors establish a method to induce spindle buckling in mid-anaphase. Time-lapse imaging shows that these spindles elongate at reduced rates, buckle as a non-homogeneous beam under mechanical load, and break at a fragile site adjacent to the midzone.These results provide quantitative and molecular insights into spindle force regulation and structural integrity that are relevant to mitosis in other cell types.

DOI: https://doi.org/10.1101/2025.11.20.689516
STAR Protoc. 2025 Nov 28. pii: S2666-1667(25)00647-1. [Epub ahead of print]6(4): 104241

Protocol for the generation of low-input Hi-C sequencing libraries of FACS-isolated mitotic cells.

Ashley Nichols, Eralda Salataj, Yujin Choi, Pierre-Jacques Hamard, Richard Koche, John Maciejowski.

  High-throughput chromosome conformation capture (Hi-C) is a powerful tool to investigate 3D genome architecture. Here, we present a protocol for preparing low-input Hi-C libraries from mitotic cells isolated by fluorescence-activated cell sorting (FACS) to study chromatin conformation in mitotic cells. We describe steps for mitotic arrest, harvest and fixation of cultured cells, staining with an anti-Mitotic Protein Marker (MPM2), and isolation of mitotic cells. We then detail procedures for quantifying input material for Hi-C in mitotic cells and library preparation of Hi-C ligated material. For complete details on the use and execution of this protocol, please refer to Nichols et al.1.

Keywords:  Cell Biology; Cell isolation; Flow Cytometry; Genomics; Molecular Biology; Sequencing

DOI:  https://doi.org/10.1016/j.xpro.2025.104241
Cell Commun Signal. 2025 Dec 04. 23(1): 519

Long-tailed class I myosins rely on tail-mediated phosphoinositide recognition for specific membrane recruitment.

Girish Rajendraprasad, Despoina Kyriazi, Peter Franz, Almke Bader, Muriel Erent, Petra Uta, Matthias Preller, Tim Scholz, Georgios Tsiavaliaris.

   BACKGROUND: Class I myosins are essential mediators of membrane-cytoskeleton interactions that support key cellular processes such as endocytosis, secretion, intracellular trafficking, and mitosis. However, the mechanisms driving isoform-specific targeting to membrane domains enriched in signaling lipids as well as their stage-dependent recruitment to mitotic structures during cell division remain poorly defined.
METHODS AND APPROACH: Using Dictyostelium discoideum as a highly phagocytic cell model, we demonstrate that long-tailed myosin-1 isoforms (myosin-1B, -1C, and - 1D) exhibit distinct lipid and cytoskeletal binding profiles shaped by their modular tails and variations within the phosphoinositide binding motif. Homology-based structural modelling of the PH-like lipid binding domain within the TH1 sequence, combined with molecular docking explains their differential lipid affinities. Kinetic equilibrium modelling with quantitative data suggests these differences enable cooperative or competitive isoform localization within cells providing a mechanism for temporally controlled recruitment of the myosins in response to dynamic changes in membrane composition and expression profiles. These biochemical insights are corroborated by confocal live-cell imaging, which reveals phosphoinositides-dependent localization dynamics and isoform-specific targeting of the myosins during vegetative growth and mitotic progression.
RESULTS: Myosin-C exhibits phosphoinositide binding preferences nearly reciprocal to those of myosin-1D, especially between mono- and triple phosphorylated phosphoinositides, and shows the strongest tail-mediated, ATP-independent actin binding. Myosin-1B, in contrast, displays low affinity for monophosphorylated phosphoinositides, intermediate actin binding ability, and no microtubule interaction. The comparable affinities of all three myosins for PI(3,5)P₂ and PI(4,5)P₂, the major PIP species at the cell cortex, facilitate their accumulation at membrane protrusions. Live-cell imaging confirms that myosin-1D preferentially associates with PI(3,4,5)P₃- and PI(3)P-enriched endosomes during macropinocytosis and phagocytosis, consistent with its higher binding affinity for these phosphoinositides. Conversely, myosin-1C localization is governed by both actin and phosphoinositides, enabling a rapid dissociation from early endosomes to retarget the cortex and accumulate at actin-rich phagocytic cup tips. Upon mitotic entry, myosin-1D, similar to myosin-1C, redistributes from endosomal compartments to the mitotic apparatus, where it decorates membrane-enclosed nuclear chromatin masses through its TH1 domain and later associates with spindle pole microtubules. This contrasts with myosin-1C, which selectively targets spindle microtubules throughout mitosis, reflecting its stronger microtubule-binding affinity. Inhibition of PI3-kinase disrupts membrane recruitment of both isoforms, confirming their phosphoinositide-dependent localization. These findings reveal an isoform-specific mechanism underlying myosin-1 targeting during endocytosis and mitosis.
CONCLUSION: Collectively, these findings establish a phosphoinositide- and cytoskeleton-guided mechanism that governs myosin-1 isoform-specific functions, providing new insights into how motor proteins interpret complex lipid and cytoskeletal cues to regulate membrane remodelling and cytoskeletal dynamics across cellular states.

Keywords:  Actin; Dictyostelium discoideum; Endocytosis; Macropinocytosis; Microtubules; Mitosis; Myosin-1; Myosin-1B; Myosin-1C; Myosin-1D; Phagocytosis; Phosphatidylinositol

DOI:  https://doi.org/10.1186/s12964-025-02528-x
J Biol Chem. 2025 Dec 01. pii: S0021-9258(25)02842-X. [Epub ahead of print] 110990

O-GlcNAcylation of the tumor suppressor LATS1 drives mitotic progression via PLK1.

Li Meng, Yunfeng Wang, Wen Zhou, Shian Wu, Jing Li.

  Initially discovered in Drosophila, the Hippo pathway is pivotal for tissue growth and organ homeostasis. It is regulated by both extrinsic and intrinsic signals and exerts its effect via a core kinase cascade, in which large tumor suppressor 1 and 2 (LATS1/2) plays a key role. LATS1 has also been shown to regulate mitotic progression by phosphorylating myosin phosphatase targeting subunit 1 (MYPT1) to counteract the activity of polo-like kinase 1 (PLK1), a mitotic master kinase. Herein we demonstrate that the hexosamine biosynthetic pathway regulates the Hippo pathway via LATS1. We show that LATS1 interacts with the O-GlcNAc transferase (OGT) and is O-GlcNAcylated. Via electron transfer dissociation mass spectrometry, we mapped the O-GlcNAcylation sites to be S479/S482/T484/T485. O-GlcNAcylation attenuates LATS1 protein stability, and downregulates the phosphorylation level of its downstream substrates, such as MYPT1. Subsequently, decreased MYPT1-pS473 levels enhance PLK1-pT210 levels and drive mitotic progression. Importantly, we demonstrate that in Drosophila O-GlcNAcylation of LATS1 promotes the wing size. Thus, this study suggests that O-GlcNAcylation links extrinsic glucose levels to LATS1 in the Hippo pathway and cell proliferation.

Keywords:  Hippo; LATS1; O-GlcNAc; OGT; PLK1

DOI:  https://doi.org/10.1016/j.jbc.2025.110990
Cancer Genet. 2025 Nov 24. pii: S2210-7762(25)00237-6. [Epub ahead of print]300-301 36-46

Quantitative and qualitative methods for measuring chromosomal instability in tumor cells.

Hanna Karolina de Araújo Batistão, João Marcos Oliveira-Silva, Vinícius Bernardo Oliveira, Isaac Iogan de Araújo Batistão, Angel Mauricio Castro-Gamero.

  To preserve cellular and tissue homeostasis, cells must maintain a coordinated network of molecular mechanisms that ensure accurate chromosome segregation during mitosis. Errors in this process, such as chromosome mis-segregation, lagging chromosomes in anaphase, centrosome amplification, or chromosome breaks generating non-homologous fusions and dicentric chromosomes, can lead to chromosomal instability (CIN). Persistent CIN promotes chromosomal abnormalities, driving tumor cell proliferation, intratumoral genomic diversity, and ultimately tumor initiation and progression. Tumors exhibiting CIN, characterized by aneuploidy or large-scale structural rearrangements, are strongly associated with metastasis, angiogenesis, and chemoresistance. Despite its biological and clinical relevance, methods to detect, quantify, and evaluate CIN remain poorly standardized. In this work, we critically reviewed the scientific literature to compile and discuss the principal quantitative and qualitative strategies currently available for measuring CIN in vitro.

Keywords:  Aneuploidy; Cancer progression; Genomic instability; Metastasis; Mitotic errors

DOI:  https://doi.org/10.1016/j.cancergen.2025.11.008
Zygote. 2025 Dec 05. 1-8

Cell cycle timing and centromere positioning in Bos taurus zygotes derived from in vitro fertilization.

Federica Modafferi, Laura Thompson, Caitríona M Collins, Elena O'Callaghan, Patrick Lonergan, Elaine M Dunleavy.

  Centromeres are chromosomal loci essential for the correct segregation of genetic material during cell division. Defects in centromere function can lead to aneuploidy and cancer. During early embryonic development in mammals, prior to the first cell division, male and female genomes are separated in pronuclei located at the centre of the zygote. Parental chromatin clusters at the interface between the two pronuclei and this clustering step is critical to avoid aneuploidy in human and bovine zygotes. Yet, despite their essential function in chromosome segregation, the position and spatial organization of centromeres during the first cell cycle in mammals is mostly unknown. Previous studies conducted in bovine embryos derived from in vitro fertilization (IVF) showed that cell cycle progression impacts on the success rate of blastocyst formation. Specifically, embryos that entered earliest into S-phase or the earliest cleaving embryos were more likely to develop into blastocysts. To determine the precise timing of these events we performed a detailed characterization of key phases of the first cell cycle in bovine zygotes derived from IVF. In parallel we examined the spatial positioning of centromeres. We identify 20 h post insemination (hpi) as the timepoint when male and female pronuclei are juxtaposed and are completing S-phase. At this timepoint, we show that centromeres are positioned distal to the pronuclear interface and use super resolution microscopy to demonstrate extensive centromere clustering into chromocentres. Our results identify distinct nuclear features observed at 20 hpi, which may serve as cell cycle markers in determining successful bovine IVF.

Keywords:  Bovine IVF; S-phase; cell cycle; centromere; chromosome; mitosis

DOI:  https://doi.org/10.1017/S0967199425100221
J Mater Chem B. 2025 Dec 01.

Lipid mapping of cell mitosis by non-covalent migratory fluorescence labelling.

Anaïs C Bourgès, Massimiliano Garre, Donal F O'Shea.

The concept of non-covalent migratory fluorescence labelling is introduced to spatially and temporally map intracellular lipids throughout a cell division cycle. This hands-off approach utilizes a small molecule BF2-azadipyrromethene fluorophore, NM-ER, to first label the nuclear membrane and endoplasmic reticulum of cells at the interphase, which can migrate with the lipid components of these structures throughout mitosis as they disassemble, redistribute and reassemble prior to daughter cell separation. Through this unique approach to image capture, key prometaphase events such as lipid intrusion into the nucleus and nuclear membrane disassembly are observable, as are the stages of nuclear membrane reassembly in the telophase and lipid distribution during cytokinesis. When used alone, NM-ER can distinguish each phase of cell mitosis from lipid staining patterns, it is compatible with STED super resolution imaging, and, with an emission maximum of 648 nm, it is usable with other common GFP and nuclear DNA stains. The non-covalent NM-ER label remains associated with the originating lipid components as they undergo architectural reorganizations and changes in subcellular localization associated with mitosis. As lipid-based cell structures are influenced by numerous biological processes and mechanical forces, our approach to fluorescence imaging could offer novel perspectives into their different roles.

DOI: https://doi.org/10.1039/d5tb01947h
Cancer Sci. 2025 Dec 05.

MASTL Promotes Hepatocellular Carcinoma Progression and Paclitaxel Resistance Through Mitotic Catastrophe.

Ke Wang, Ziwei Fu, Chuan Xiong, Jing Zhang, Qiang Luo.

  Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with limited therapeutic options. Microtubule-associated serine/threonine kinase-like (MASTL), a pivotal regulator of mitosis, remains poorly characterized in HCC. This study aimed to elucidate the clinical significance, biological functions, and molecular mechanisms of MASTL in HCC progression. Bioinformatics analysis of TCGA and ICGC datasets revealed MASTL overexpression correlated with advanced tumor stage and served as an independent prognostic factor. Functional studies demonstrated that MASTL knockdown significantly disrupts HCC cell proliferation, increases the incidence of abnormal mitotic events, and amplifies DNA damage, collectively driving mitotic catastrophe (MC) and subsequent cell death. Mechanistically, MASTL regulated paclitaxel sensitivity by modulating ENSA phosphorylation and PP2A-B55α activity, with PP2A-B55α knockdown reversing MASTL deficiency-induced MC. Transcriptional regulation analysis identified E2F1 as a direct activator of MASTL expression, confirmed by ChIP-qPCR and dual-luciferase reporter assays. These findings establish MASTL as a critical oncogene in HCC through the E2F1-MASTL-PP2A-B55α axis, suggesting its potential as both a prognostic biomarker and therapeutic target for HCC. Future studies should explore MASTL inhibitors in combination with conventional chemotherapy to overcome drug resistance in HCC patients.

Keywords:  DNA damage; MASTL; greatwall kinase; hepatocellular carcinoma; mitotic catastrophe

DOI:  https://doi.org/10.1111/cas.70287
Methods. 2025 Dec 01. pii: S1046-2023(25)00241-5. [Epub ahead of print]

Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and cell cycle progression.

Romina Ines Cervigni, Raffaella Bonavita, Maria Luisa Barretta, Daniela Spano, Inmaculada Ayala, Fabiola Mascanzoni, Roberta Iannitti, Petra Henklein, Alessandra Monti, Maurizio Renna, Nunzianna Doti, Antonino Colanzi.

  The Golgi complex is central to cellular homeostasis and serves as a key processing and sorting hub for protein trafficking. In many cell types, the Golgi complex is organized as interconnected stacks of cisternae, forming a structure known as the Golgi ribbon. This ribbon undergoes dynamic remodelling during physiological processes, such as cell division, and under pathological conditions, including cancer and neurodegeneration. A critical step in the unlinking of the Golgi ribbon involves the phosphorylation of the stacking protein GRASP65, which leads to the separation of the ribbon into individual stacks, a process necessary for the G2/M transition of the cell cycle. However, existing tools for selectively manipulating the GRASP65 role in ribbon organization are limited by non-specific effects or technical challenges. Here, we present the development and characterization of a membrane-permeable peptide, R8-GRASP65-S277, derived from GRASP65 and containing the phosphorylation site Ser277, which is essential for Golgi unlinking. This peptide effectively inhibited Golgi unlinking and mitotic entry in several cell lines, including cancer models. In contrast, a control peptide with a non-phosphorylatable alanine substitution (R8-GRASP65-S277A) showed no such effect, confirming the specificity of the tool. Furthermore, the R8-GRASP65-S277 peptide reversed Golgi unlinking induced by the chemotherapeutic agent doxorubicin, demonstrating its utility in studying stress-induced Golgi disassembly. These findings establish the R8-GRASP65-S277 peptide as a specific, potent, and scalable tool for probing the molecular mechanisms of Golgi unlinking, its regulation of cell cycle progression, and its potential contributions to pathological states.

Keywords:  Cell cycle checkpoint; GRASP65; Golgi complex; Golgi unlinking; Membrane-permeant peptide

DOI:  https://doi.org/10.1016/j.ymeth.2025.11.008