bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2024–12–08
nineteen papers selected by
Valentina Piano, Uniklinik Köln
  1. Super-Resolution Imaging of Mitotic Spindle Microtubules Using STED Microscopy.
  2. CCDC69 maintains genome integrity by regulating KIF2C/MCAK depolymerase activity and the stability of the chromosomal passenger complex.
  3. 4D Microscopy and Tracking of Chromosomes and the Spindle in C. elegans Early Embryos.
  4. A Method for Analyzing Acentrosomal Mitotic Spindles in Human Cells.
  5. Isolation of Mitotic Centrosomes from Cultured Human Cells.
  6. Force generation and resistance in human mitosis.
  7. Assessment of Chromosome Oscillations in Mammalian Cells by Live Cell Imaging.
  8. Molecular details and phosphoregulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells.
  9. Manipulation of Spindle Position Using Magnetic Tweezers in Sea Urchin Embryos.
  10. Synthetic Engineering of Cortical Polarity During Mitosis Using Designed Proteins.
  11. Photochemical Control of Cell Division Using a Photoswitchable CENP-E Inhibitor.
  12. HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis.
  13. TNK2 Inhibitor (R)-9bMS Causes Polyploidization Through Mitotic Failure by Targeting Aurora B.
  14. Spatial Statistics of Three-Dimensional Growth Dynamics of Spindle Microtubules.
  15. The kinetochore protein KNL-1 regulates the actin cytoskeleton to control dendrite branching.
  16. Time-Lapse Imaging of Asymmetric Spindle Positioning During Endothelial Tip Cell Division in Angiogenesis In Vivo.
  17. Phosphorylation of Lamin A/C regulates structural integrity of the nuclear envelope.
  18. Proteome-wide forced interactions reveal a functional map of cell-cycle phospho-regulation in S. cerevisiae.
  19. Cell Division Imaging of Tobacco BY-2 Cells in a 3D Volume by Two-Photon Spinning-Disk Confocal Microscopy.
  1. Methods Mol Biol. 2025 ;2872 3-19
    Super-Resolution Imaging of Mitotic Spindle Microtubules Using STED Microscopy.
    Isabella Koprivec, Valentina Štimac, Iva M Tolić.
      Stimulated emission depletion (STED) microscopy is a powerful super-resolution imaging technique that only recently entered the field of mitosis, where it proved to be invaluable for studying various microtubule classes, kinetochore-microtubule attachments and chromosome segregation errors. Here, we describe immunofluorescence combined with STED microscopy as a method for analyzing microtubules and kinetochore-microtubule attachments in human mitotic spindles. We also describe live-cell STED microscopy as a method for single-plane short-term imaging of transient processes in crowded spindle areas. Finally, we outline image analysis approaches for the quantitative assessment of microtubule bundles within the spindle.
    Keywords:  Attachments; Cell division; Chromosomes; Kinetochores; Microtubules; Mitosis; Mitotic spindle; Nucleation; STED microscopy; Segregation errors
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_1
  2. Sci Rep. 2024 Dec 05. 14(1): 30401
    CCDC69 maintains genome integrity by regulating KIF2C/MCAK depolymerase activity and the stability of the chromosomal passenger complex.
    Didier Fesquet, Gabriel Rabeharivelo, Juliette van Dijk, Claude Prigent, Nathalie Morin, Sylvie Rouquier.
      Accurate genome inheritance during cell division relies on a complex chromosome segregation mechanism. This process occurs once all the kinetochores of sister chromatids are attached to microtubules emanating from the opposite poles of the mitotic spindle. To control the precision of this mechanism, the Chromosome Passenger Complex (CPC) actively identifies and corrects improper microtubule attachments. The depolymerase activity of the kinesin KIF2C/MCAK at the kinetochores is involved in this process. CCDC69 is a poorly characterized protein, primarily identified as a regulator of central spindle assembly, whose overexpression prompts rapid microtubule depolymerization. Here, we show that CCDC69 is a cell-cycle regulated protein belonging to the Microtubule-associated Tumor Suppressor (MTUS) superfamily, and even slight deregulation of its expression induces severe early mitotic phenotypes. Myristoylation anchors CCDC69 at the plasma membrane, thus protecting microtubule network integrity. We found that CCDC69 microtubule depolymerization activity relies on KIF2C, with a fraction of CCDC69 localizing to the centromere. Importantly, we demonstrated that CCDC69 regulates the stability of the CPC by safeguarding its members from degradation during mitosis. In summary, our findings underscore CCDC69's essential role as a mitotic regulator, which is crucial for maintaining the fidelity of chromosome segregation.
    Keywords:  CCDC69; Chromosomal Passenger Complex; KIF2C/MCAK; Microtubule dynamics; Mitosis; Myristoylation
    DOI:  https://doi.org/10.1038/s41598-024-81022-9
  3. Methods Mol Biol. 2025 ;2872 141-156
    4D Microscopy and Tracking of Chromosomes and the Spindle in C. elegans Early Embryos.
    Julien Dumont, Gilliane Maton.
      Maintaining genomic integrity throughout successive cell divisions is essential for the proper development and functioning of organisms. Chromosome alignment and segregation occur on a microtubule-based spindle originating from centrosomes. The molecular and cellular mechanisms involved in accurate chromosome segregation during early embryonic divisions are highly conserved between worms and humans. Therefore, C. elegans serves as a robust model for investigating mitotic cell divisions within a metazoan system. Throughout early embryonic development, filming and tracking successive cell divisions becomes progressively more challenging as the number of cells increases and cell size decreases. To address this challenge, we describe a method for preparing live samples, performing 4D time-lapse imaging, and semi-automated tracking of chromosomes and spindle poles during early mitotic divisions in C. elegans embryos.
    Keywords:  C. elegans; Cell division; Chromosome segregation; Embryonic division; Mitosis; Mitotic spindle; Time-lapse imaging
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_10
  4. Methods Mol Biol. 2025 ;2872 221-231
    A Method for Analyzing Acentrosomal Mitotic Spindles in Human Cells.
    Shotaro Okuda, Takumi Chinen, Daiju Kitagawa.
      Centrosomes, the major microtubule organizing centers, facilitate mitotic spindle formation. However, recent studies have revealed that some cancer cells lack centrosomes. These findings suggest that certain types of cancer cells drive centrosome-independent mechanisms for the assembly of mitotic spindles. Therefore, analyzing the systems of acentrosomal spindle formation is beneficial for understanding the divergence of mitotic processes in cancer cells. Here, we focus on a method for generating acentrosomal cells using the PLK4 inhibitor centrinone and describe the proper conditions, spindle pole staining markers, and microscopy settings for observing acentrosomal spindle formation in human cells.
    Keywords:  Acentrosomal spindle; Centrinone; Centrosome; NuMA; Spindle pole
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_15
  5. Methods Mol Biol. 2025 ;2872 233-244
    Isolation of Mitotic Centrosomes from Cultured Human Cells.
    Momoko Miyazawa, Shohei Yamamoto, Susumu Goyama, Daiju Kitagawa.
      The centrosome plays a crucial role in facilitating mitotic spindle assembly through its microtubule organizing capacities. Analyzing the composition, structure, and functions of mitotic centrosomes is essential for understanding the mechanisms underlying cell division and centrosome-associated diseases. Isolating centrosomes is an effective method to gain comprehensive information about them while minimizing interference from other cellular components. In this chapter, we describe a protocol for isolating mitotic centrosomes from cultured human cells. This protocol includes cell synchronization and centrosome isolation through ultracentrifugation with a sucrose gradient. We also describe a method for conducting a microtubule nucleation assay to assess the functionality of isolated centrosomes.
    Keywords:  Centriole; Centrosome; MTOC; Microtubule; Mitosis; PCM; Sucrose gradient
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_16
  6. Biophys Rev. 2024 Oct;16(5): 551-562
    Force generation and resistance in human mitosis.
    Colleen C Caldwell, Tinka V M Clement, Gijs J L Wuite.
      Since the first observations of chromosome segregation over 150 years ago, efforts to observe the forces that drive mitosis have evolved alongside advances in microscopy. The mitotic spindle acts as the major generator of force through the highly regulated polymerization and depolymerization of microtubules as well as associated motor proteins. Centromeric chromatin, along with associated proteins including cohesin and condensin, is organized to resist these forces and ensure accurate chromosome segregation. Microtubules and centromeric chromatin join at the kinetochore, a complex protein superstructure. Ongoing research into the forces generated at the kinetochore-microtubule interface has resulted in a range of estimates for forces necessary to separate chromosomes, from tens to hundreds of piconewtons. Still, the exact magnitude and regulation of these forces remain areas of continuing investigation. Determining the precise forces involved in chromosome segregation is hindered by limitations of current measurement techniques, but advances such as optical tweezers combined with fluorescence microscopy are promising for future research.
    Keywords:  Centromere; Chromosome; Mitosis; Spindle
    DOI:  https://doi.org/10.1007/s12551-024-01235-0
  7. Methods Mol Biol. 2025 ;2872 157-164
    Assessment of Chromosome Oscillations in Mammalian Cells by Live Cell Imaging.
    Kenji Iemura, Kozo Tanaka.
      In metaphase, chromosomes undergo a back-and-forth movement between two spindle poles called chromosome oscillation. This dynamic is necessary to maintain the robustness of chromosome segregation. This chapter describes the materials and methods required to observe chromosome oscillation in mammalian cell lines and calculate the deviation of amplitude (DAP), which is used to quantify the degree of chromosome oscillation.
    Keywords:  Chromosome dynamics; Kinetochore; Metaphase; Microscope; Mitotic spindle; Time-lapse imaging
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_11
  8. iScience. 2024 Dec 20. 27(12): 111295
    Molecular details and phosphoregulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells.
    Yusuke Takenoshita, Masatoshi Hara, Reiko Nakagawa, Mariko Ariyoshi, Tatsuo Fukagawa.
      To establish bipolar attachments of microtubules to sister chromatids, an inner kinetochore subcomplex, the constitutive centromere-associated network (CCAN), is assembled on centromeric chromatin and recruits the microtubule-binding subcomplex called the KMN network. Since CCAN proteins CENP-C and CENP-T independently bind to the Mis12 complex (Mis12C) of KMN, it is difficult to evaluate the significance of each interaction in cells. Here, we demonstrate the molecular details of the CENP-T-Mis12C interaction using chicken DT40 cells lacking the CENP-C-Mis12C interaction. Using AlphaFold predictions combined with cell biological and biochemical analyses, we identified three binding surfaces of the CENP-T-Mis12C interaction, demonstrating that each interface is important for recruiting Mis12C to CENP-T in cells. This interaction, via three interaction surfaces, is cooperatively regulated by dual phosphorylation of Dsn1 (a Mis12C component) and CENP-T, ensuring a robust CENP-T-Mis12C interaction and proper mitotic progression. These findings deepen our understanding of kinetochore assembly in cells.
    Keywords:  Biological sciences; Biophysics; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2024.111295
  9. Methods Mol Biol. 2025 ;2872 87-100
    Manipulation of Spindle Position Using Magnetic Tweezers in Sea Urchin Embryos.
    Aude Nommick, Jing Xie, Nicolas Minc.
      The regulation of mitotic spindle position and orientation, and consequent division plane specification, is critical for early embryo development, tissue architecture and stem cells. Accordingly, defects in spindle positioning have been associated with the emergence of tissue disorders and mis-specification of cell fates, causing the formation of tumors, for instance, in stem cell populations. In such context, methods to physically manipulate mitotic spindle position or orientation in living cells bear the promise of dissecting the causal impact of spindle mis-positioning on cell or tissue behavior. Here, we describe a method to directly modulate mitotic spindle position and orientation with in vivo magnetic tweezers in developing sea urchin embryos. This method allows for monitoring the impact of spindle orientation on embryo development and quantifying forces and torques needed to move or rotate spindles. It may be transposable to many other cell, embryo or tissue types.
    Keywords:  Cell division; Embryogenesis; Forces; Magnetic tweezers; Microinjection; Mitosis; Morphogenesis; Sea urchin; Spindle
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_6
  10. Methods Mol Biol. 2025 ;2872 247-255
    Synthetic Engineering of Cortical Polarity During Mitosis Using Designed Proteins.
    Lara K Krüger, Joseph L Watson, Emmanuel Derivery.
      During asymmetric cell division, cortical polarity cues drive the polarization of the microtubule cytoskeleton to ensure the generation of two daughter cells with different fates. While this a critical process for development and tissue homeostasis, the underlying molecular mechanisms orchestrating those processes are not completely understood, especially in mammals. Here, we present an assay that allows the study of the molecular mechanisms driving mammalian asymmetric cell division in a high-throughput manner by capitalizing on protein design to engineer cortical polarity of virtually any protein of interest in otherwise unpolarized mammalian culture cells.
    Keywords:  Asymmetric cell division; Cell polarity; Microtubule; Mitotic spindle; Synthetic biology
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_17
  11. Methods Mol Biol. 2025 ;2872 101-113
    Photochemical Control of Cell Division Using a Photoswitchable CENP-E Inhibitor.
    Akira Matsura, Shota Hiruma, Nobuyuki Tamaoki, Ryota Uehara.
      Photoswitchable compounds are potent tools for elucidating molecular functions in dynamic cellular processes. Photoswitchable inhibitors targeting various mitotic spindle factors have been developed. In this chapter, we describe experimental methods for photo-controlling mitotic chromosome dynamics using a recently developed photoswitchable inhibitor of mitotic kinesin, CENP-E. This inhibitor undergoes reversible photoisomerization to a more inhibitory trans or less inhibitory cis state by visible or UV light irradiation, respectively, enabling photoswitching of CENP-E function both in vitro and in vivo. First, we explain the procedures used to optimize the experimental condition for efficient photoswitching of CENP-E functionality in cultured cells. We then describe how to conduct de novo photo-control of mitotic chromosome motion using the inhibitor under a microscope.
    Keywords:  Azobenzene; Chromosome segregation; Mitotic kinesin; Photoswitch
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_7
  12. J Virol. 2024 Dec 04. e0093724
    HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis.
    Timothy R Keiffer, Stephen DiGiuseppe, Lucile Guion, Malgorzata Bienkowska-Haba, Katarzyna Zwolinska, Abida Siddiqa, Anand Kushwaha, Martin J Sapp.
      Human papillomaviruses (HPVs) travel from the trans-Golgi network (TGN) to the condensed (mitotic) chromosomes during mitosis. Partially uncoated HPV capsids utilize a unique vesicular structure for trafficking and nuclear import, which is directed by the minor capsid protein L2. However, it is still unknown which precise factors facilitate post-TGN HPV trafficking to the nucleus. Herein, we analyzed HPV16-infected mitotic cells using high-resolution microscopy, coupled with motor protein inhibition, to further elaborate on post-TGN trafficking by tracking the location and/or quantification of EdU-labeled HPV pseudogenomes on microtubules, certain kinesins, and mitotic chromosomes. We also adapted a knocksideways approach to determine if L2 and Kif11 interact in infected cells. We visualized dynein co-localization with HPV pseudogenomes along mitotic microtubules and measured HPV pseudogenome accumulation after short-term dynein inhibition. Additional inhibitor studies implicated a specific kinesin, Kif11, as participating in HPV pseudogenome delivery to the nucleus. Short-term inhibition of Kif11 decreased HPV pseudogenome accumulation at mitotic chromatin. In addition, Kif11, along with kinesins Kif18a and Kif25, were in proximity to L2 during infection. While we were unable to determine a direct interaction between L2 and Kif11, we were able to show via knocksideways approach that relocalization of exogenous Kif11 decreased HPV pseudogenome accumulation to the mitotic chromatin. Our data support a model whereby HPV16 utilizes dynein for minus-end trafficking along mitotic microtubules and utilizes Kif11 for plus-end movement in the late stage of viral entry.
    IMPORTANCE: Human papillomaviruses (HPV) utilize a unique vesicular structure to shield their genomes from detection during trafficking from the trans-Golgi network (TGN) to the nucleus during mitosis. The exact cellular factors responsible for trafficking these HPV genome containing vesicles along mitotic microtubules via the L2 minor protein remain unknown. We show via high-resolution microscopy that pharmacological inhibition of dynein and the kinesin Kif11 significantly decreases HPV pseudogenome accumulation on mitotic chromatin. Several kinesins were detected in proximity to incoming HPV pseudogenomes. Finally, using a novel knocksideways approach, we show reduced HPV pseudogenome accumulation on mitotic chromatin upon Kif11 relocalization to the mitochondria. Herein, our data suggest HPV utilizes minus- and plus-end mediated trafficking along mitotic microtubules to complete its genome trafficking to the nucleus.
    Keywords:  HPV16; Kif11; Kif18a; Kif25; dynein; kinesins; knocksideways; microtubules; mitosis; proximity ligation assay
    DOI:  https://doi.org/10.1128/jvi.00937-24
  13. Cell Biochem Funct. 2024 Dec;42(8): e70022
    TNK2 Inhibitor (R)-9bMS Causes Polyploidization Through Mitotic Failure by Targeting Aurora B.
    Mayu Murata, Hiroki Kuwajima, Junna Tanaka, Nanami Hasegawa, Ryuzaburo Yuki, Youhei Saito, Yuji Nakayama.
      TNK2 is a ubiquitously expressed nonreceptor-type tyrosine kinase. TNK2 participates in tumorigenesis, and TNK2 activation has been found in various cancers; therefore, TNK2 is a promising target for cancer chemotherapy. While the TNK2 inhibitor XMD16-5 is highly selective, it inhibits cytokinesis at higher concentrations by targeting Aurora B kinase, a key enzyme for cell division. Cytokinesis failure frequently generates polyploid cells, and the surviving polyploid cells risk leading to cancer development and malignant progression via chromosome instability. In this study, to investigate the possibility that (R)-9bMS, a TNK2 inhibitor structurally related to XMD16-5, drives malignant progression by inducing abnormal cell division, we examined its effects on cell division, Aurora B autophosphorylation, and colony formation. Cell count results showed a reduction in the number of A431, HeLa S3, HCT116, and MCF7 cells upon TNK2 inhibitor treatment. Microscopic observation indicated the formation of multinucleated and nucleus-enlarged cells. An increase in DNA content was confirmed with flow cytometry, which was underpinned by an increased number of centrosomes. Time-lapse imaging revealed mitotic failure, such as mitotic slippage and cytokinesis failure, as a cause of polyploidization. Of note, TNK2 knockdown significantly increased multinucleated cells, but the effect was quite weak, suggesting that TNK2 inhibition may only partially contribute to mitotic failure and polyploidization. Expectedly, Aurora B phosphorylation was reduced by (R)-9bMS like XMD16-5, but not by TNK2 knockdown. Collectively, TNK2 inhibitors (R)-9bMS and XMD16-5 induce polyploidization via mitotic failure caused by the inhibition of Aurora B kinase rather than TNK2. Notably, (R)-9bMS treatment promoted anchorage-independent colony formation, a hallmark of cancer. Our findings suggest that (R)-9bMS at a high concentration risks promoting cancer development or malignant progression. Therefore, caution should be used when using TNK2 inhibitors for cancers where TNK2 activation is not the transforming mutation and higher concentrations of TNK2 inhibitors are required to slow proliferation.
    Keywords:  Aurora B; TNK2; cell division; cytokinesis; mitotic slippage; polyploidization
    DOI:  https://doi.org/10.1002/cbf.70022
  14. Methods Mol Biol. 2025 ;2872 51-72
    Spatial Statistics of Three-Dimensional Growth Dynamics of Spindle Microtubules.
    Norio Yamashita, Masahiko Morita, Hideo Yokota, Yuko Mimori-Kiyosue.
      The latest high-resolution 3D live-cell imaging technology, lattice light-sheet microscopy (LLSM), has successfully tracked the dynamics of microtubule growth throughout the entire mitotic spindle with unparalleled precision. By using green fluorescent protein-labeled end-binding protein 1 (EB1-GFP) as a marker for growing microtubule ends, LLSM has generated an extensive collection of multidimensional datasets mapping the positions and trajectories of these growing microtubule ends. Processing this data requires statistical analysis in three-dimensional space. This chapter describes the spatial statistical methods developed for this purpose, illustrated with practical examples. Finally, we discuss future prospects for analyzing complex, large-scale image data.
    Keywords:  EB1; LLSM; Lattice light-sheet microscopy; Microtubule dynamics; Mitosis; Spatial statistics; Spindle
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_4
  15. J Cell Biol. 2025 Feb 03. pii: e202311147. [Epub ahead of print]224(2):
    The kinetochore protein KNL-1 regulates the actin cytoskeleton to control dendrite branching.
    Henrique Alves Domingos, Mattie Green, Vasileios R Ouzounidis, Cameron Finlayson, Bram Prevo, Dhanya K Cheerambathur.
      The function of the nervous system is intimately tied to its complex and highly interconnected architecture. Precise control of dendritic branching in individual neurons is central to building the complex structure of the nervous system. Here, we show that the kinetochore protein KNL-1 and its associated KMN (Knl1/Mis12/Ndc80 complex) network partners, typically known for their role in chromosome-microtubule coupling during mitosis, control dendrite branching in the Caenorhabditis elegans mechanosensory PVD neuron. KNL-1 restrains excess dendritic branching and promotes contact-dependent repulsion events, ensuring robust sensory behavior and preventing premature neurodegeneration. Unexpectedly, KNL-1 loss resulted in significant alterations of the actin cytoskeleton alongside changes in microtubule dynamics within dendrites. We show that KNL-1 modulates F-actin dynamics to generate proper dendrite architecture and that its N-terminus can initiate F-actin assembly. These findings reveal that the postmitotic neuronal KMN network acts to shape the developing nervous system by regulating the actin cytoskeleton and provide new insight into the mechanisms controlling dendrite architecture.
    DOI:  https://doi.org/10.1083/jcb.202311147
  16. Methods Mol Biol. 2025 ;2872 269-286
    Time-Lapse Imaging of Asymmetric Spindle Positioning During Endothelial Tip Cell Division in Angiogenesis In Vivo.
    Holly E Lovegrove, Shane P Herbert.
      The branching of new blood vessels by angiogenesis is critical to the development, growth, and repair of most vertebrate tissues and is frequently dysregulated in disease. At its core, angiogenesis is driven by the collective migration of leading "tip" and follower "stalk" endothelial cells. Recent work reveals that this collective movement is coordinated by asymmetric tip cell divisions that generate daughters of distinct size, signaling capacity and tip-stalk behaviors. Polarized mitotic spindle positioning is critical to generating such asymmetries in daughter cell size. However, the spatiotemporal dynamics of vertebrate spindle movement are often difficult to explore using in vivo systems. Here we describe a method for the sample preparation, live-imaging and data analysis of endothelial cell mitotic spindle positioning in developing zebrafish embryos. This method enables single-cell and population-level spindle dynamics to be monitored and quantified, both in wild-type or genetically/pharmacologically perturbed embryos. Moreover, this approach can be easily adapted for live imaging of spindle dynamics in other zebrafish embryonic tissues that experience similar asymmetric divisions, such as the trunk neural crest.
    Keywords:  Angiogenesis; Endothelial; Live imaging; Spindle; Transgenic; Tubulin; Zebrafish
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_19
  17. J Biol Chem. 2024 Nov 28. pii: S0021-9258(24)02535-3. [Epub ahead of print] 108033
    Phosphorylation of Lamin A/C regulates structural integrity of the nuclear envelope.
    Shuaiyu Liu, Fangyuan Xiong, Zhen Dou, Lingluo Chu, Yihan Yao, Ming Wang, Xuebiao Yao, Xing Liu, Zhikai Wang.
      Dynamic disassembly and reconstruction of the nuclear lamina during entry and exit of mitosis, respectively, are pivotal steps in proliferation of higher eukaryotic cells. Although numerous post-translational modifications of lamin proteins have been identified, key factors driving the nuclear lamina dynamics remain elusive. Here we identified CDK1-elicited phosphorylation sites on endogenous Lamin A/C and characterized their functions in regulation of the nuclear lamina. Specifically, mass spectrometry revealed CDK1-mediated phosphorylation of Lamin A/C at the N-terminal Thr19/Ser22 and the C-terminal Ser390/Ser392 during mitosis. Importantly, the phospho-mimicking 4D mutant T19D/S22D/S390D/S392D completely disrupted Lamin A filamentous structure in interphase cells. Conversely, the non-phosphorylatable mutant T19A/S22A and especially the 4A mutant T19A/S22A/S390A/S392A protected Lamin A from depolymerization during mitosis. These results suggest that phosphorylation and dephosphorylation of both N- and C-terminal sites regulate the nuclear lamina dynamics. Engineering the non-phosphorylatable mutant T19A/S22A into the endogenous LMNA gene resulted in nuclear abnormalities and micronucleus formation during telophase. Perturbation of the Lamin A phosphorylation is shown to prevent proper nuclear envelope dynamics and impair nuclear integrity. These findings reveal a previously undefined link between the CDK1-elicited Lamin A phosphorylation dynamics, nuclear envelope plasticity, and genomic stability during the cell cycle.
    Keywords:  CDK1; Lamin A; mitosis; nuclear integrity; phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2024.108033
  18. Nucleus. 2024 Dec;15(1): 2420129
    Proteome-wide forced interactions reveal a functional map of cell-cycle phospho-regulation in S. cerevisiae.
    Cinzia Klemm, Guðjón Ólafsson, Henry Richard Wood, Caitlin Mellor, Nicolae Radu Zabet, Peter Harold Thorpe.
      Dynamic protein phosphorylation and dephosphorylation play an essential role in cell cycle progression. Kinases and phosphatases are generally highly conserved across eukaryotes, underlining their importance for post-translational regulation of substrate proteins. In recent years, advances in phospho-proteomics have shed light on protein phosphorylation dynamics throughout the cell cycle, and ongoing progress in bioinformatics has significantly improved annotation of specific phosphorylation events to a given kinase. However, the functional impact of individual phosphorylation events on cell cycle progression is often unclear. To address this question, we used the Synthetic Physical Interactions (SPI) method, which enables the systematic recruitment of phospho-regulators to most yeast proteins. Using this method, we identified several putative novel targets involved in chromosome segregation and cytokinesis. The SPI method monitors cell growth and, therefore, serves as a tool to determine the impact of protein phosphorylation on cell cycle progression.
    Keywords:  CDK; Cdc5; Cdc7; cell cycle; phosphatases; phospho-regulation; synthetic physical interactions
    DOI:  https://doi.org/10.1080/19491034.2024.2420129
  19. Methods Mol Biol. 2025 ;2872 37-50
    Cell Division Imaging of Tobacco BY-2 Cells in a 3D Volume by Two-Photon Spinning-Disk Confocal Microscopy.
    Takashi Murata, Motosuke Tsutsumi, Kohei Otomo, Tomomi Nemoto.
      Plant cells have a unique organization of microtubules during mitosis and cytokinesis. However, it is difficult to observe cell division in plant cells because of the distortion of images caused by thick samples. We addressed this issue by two-photon excitation coupled with spinning-disk confocal microscopy. Herein, we describe the method for observing microtubules and nuclei during cell division in tobacco BY-2 cells by two-photon spinning-disk confocal microscopy. The protocol includes the transformation of BY-2 cells with a plasmid for the expression of tubulin and histone markers, preparation of cells for microscopy, and operation of the two-photon spinning-disk microscope.
    Keywords:  Cell division; Microtubule; Spinning disk confocal microscopy; Two-photon excitation
    DOI:  https://doi.org/10.1007/978-1-0716-4224-5_3
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