bims-micesi 2023-07-16 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2023–07–16
eleven papers selected by
Valentina Piano, Uniklinik Köln

FOXM1 is critical for the fitness recovery of chromosomally unstable cells.
Misregulation of cell cycle dependent methylation of budding yeast CENP-A contributes to chromosomal instability.
3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM).
RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells.
Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System.
Oiling the wheels of nuclear division: SUMOylation regulates the expansion of the mitotic nuclear membrane.
Asymmetric mitosis contributes to different migratory performance in sister cells.
Centripetal nuclear shape fluctuations associate with chromatin condensation in early prophase.
Polarized SCAR and the Arp2/3 complex regulate apical cortical remodeling in asymmetrically dividing neuroblasts.
The Crucial Role of CTCF in Mitotic Progression during Early Development of Sea Urchin.
MCAK Inhibitors Induce Aneuploidy in Triple-Negative Breast Cancer Models.

Cell Death Dis. 2023 Jul 14. 14(7): 430

FOXM1 is critical for the fitness recovery of chromosomally unstable cells.

Fan Pan, Sara Chocarro, Maria Ramos, Yuanyuan Chen, Alicia Alonso de la Vega, Kalman Somogyi, Rocio Sotillo.

Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein MAD2, leading to mitotic arrest and cell death. However, some tumor cells are capable of exiting mitosis and consequently increasing CIN. How cells escape the mitotic arrest induced by MAD2 and proliferate with CIN is not well understood. Here, we explored loss-of-function screens and drug sensitivity tests associated with MAD2 levels in aneuploid cells and identified that aneuploid cells with high MAD2 levels are more sensitive to FOXM1 depletion. Inhibition of FOXM1 promotes MAD2-mediated mitotic arrest and exacerbates CIN. Conversely, elevating FOXM1 expression in MAD2-overexpressing human cell lines reverts prolonged mitosis and rescues mitotic errors, cell death and proliferative disadvantages. Mechanistically, we found that FOXM1 facilitates mitotic exit by inhibiting the spindle assembly checkpoint (SAC) and the expression of Cyclin B. Notably, we observed that FOXM1 is upregulated upon aneuploid induction in cells with dysfunctional SAC and error-prone mitosis, and these cells are sensitive to FOXM1 knockdown, indicating a novel vulnerability of aneuploid cells.

DOI: https://doi.org/10.1038/s41419-023-05946-2
Mol Biol Cell. 2023 Jul 12. mbcE23030108

Misregulation of cell cycle dependent methylation of budding yeast CENP-A contributes to chromosomal instability.

Prashant K Mishra, Wei-Chun Au, Pedro G Castineira, Nazrin Ali, John Stanton, Lars Boeckmann, Yoshimitsu Takahashi, Michael Costanzo, Charles Boone, Kerry S Bloom, Peter H Thorpe, Munira A Basrai.

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle regulated methylation of Cse4 in high fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.

DOI: https://doi.org/10.1091/mbc.E23-03-0108
Bio Protoc. 2023 Jul 05. 13(13): e4708

3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM).

Nuria Ferrandiz, Stephen J Royle.

  Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides high-resolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.

Keywords:  Chromosome missegregation; Endoplasmic reticulum; Microscopy; Mitosis; Scanning electron microscopy; Volume EM

DOI:  https://doi.org/10.21769/BioProtoc.4708
EMBO Rep. 2023 Jul 10. e56240

RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells.

Ivor Joseph, Juan Flores, Victoria Farrell, Justin Davis, Jared Bianchi-Smak, Qiang Feng, Sayantani Goswami, Xiang Lin, Zhi Wei, Kevin Tong, Zhaohui Feng, Michael P Verzi, Edward M Bonder, James R Goldenring, Nan Gao.

  RAB11 small GTPases and associated recycling endosome have been localized to mitotic spindles and implicated in regulating mitosis. However, the physiological significance of such regulation has not been observed in mammalian tissues. We have used newly engineered mouse models to investigate intestinal epithelial renewal in the absence of single or double isoforms of RAB11 family members: Rab11a and Rab11b. Comparing with single knockouts, mice with compound ablation demonstrate a defective cell cycle entry and robust mitotic arrest followed by apoptosis, leading to a total penetrance of lethality within 3 days of gene ablation. Upon Rab11 deletion ex vivo, enteroids show abnormal mitotic spindle formation and cell death. Untargeted proteomic profiling of Rab11a and Rab11b immunoprecipitates has uncovered a shared interactome containing mitotic spindle microtubule regulators. Disrupting Rab11 alters kinesin motor KIF11 function and impairs bipolar spindle formation and cell division. These data demonstrate that RAB11A and RAB11B redundantly control mitotic spindle function and intestinal progenitor cell division, a mechanism that may be utilized to govern the homeostasis and renewal of other mammalian tissues.

Keywords:  KIF11; RAB11A; RAB11B; mitosis; spindle

DOI:  https://doi.org/10.15252/embr.202256240
J Vis Exp. 2023 06 23.

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System.

Meng-Fei Xu, Jie Chen, Yue Xu, Jing-Lian Zhang, Yi Zhou, Jie-Jie He, Shan Wu, Ya-Lan Wei, Zhen-Yu She.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors. In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

DOI: https://doi.org/10.3791/65476
J Cell Biol. 2023 Aug 07. pii: e202306126. [Epub ahead of print]222(8):

Oiling the wheels of nuclear division: SUMOylation regulates the expansion of the mitotic nuclear membrane.

Symeon Siniossoglou.

Eukaryotic cell division involves the segregation of chromosomes between two daughter cells and must be coordinated with extensive rearrangement of their nuclear envelopes. In this issue, Saik et al. (2023 J. Cell Biol. https://doi.org/10.1083/jcb.202208137) show that a SUMOylation cascade at the inner nuclear membrane elevates the levels of phosphatidic acid, a key phospholipid precursor, to support the need for nuclear membrane expansion during mitosis.

DOI: https://doi.org/10.1083/jcb.202306126
Exp Cell Res. 2023 Jul 08. pii: S0014-4827(23)00263-X. [Epub ahead of print] 113715

Asymmetric mitosis contributes to different migratory performance in sister cells.

Maurício Tavares Tamborindeguy, Paola Farias Lorenzatto, Marcelo Lazzaron Lamers, Guido Lenz.

  In cancer, cell migration contributes to the spread of tumor cells resulting in metastasis. Heterogeneity in the migration capacity can produce individual cells with heightened capacity leading to invasion and metastasis. Our hypothesis is that cell migration characteristics can divide asymmetrically in mitosis, allowing a subset of cells to have a larger contribution to invasion and metastasis. Therefore, our aim is to elucidate whether sister cells have different migratory capacity and analyze if this difference is defined by mitosis. Through time-lapse videos, we analyzed migration speed, directionality, maximum displacement of each trajectory, and velocity as well as cell area and polarity and then compared the values between mother-daughter cells and between sister cells of three tumor cell lines (A172, MCF7, SCC25) and two normal cell lines (MRC5 and CHO·K1 cells). We observed that daughter cells had a different migratory phenotype compared to their mothers, and one single mitosis is enough for the sisters behave like nonrelated cells. However, mitosis did not influence cell area and polarity dynamics. These findings indicates that migration performance is not heritable, and that asymmetric cell division might have an important impact on cancer invasion and metastasis, by producing cells with different migratory capacity.

Keywords:  Asymmetric mitosis; Cell heterogeneity; Cell migration; Single cell

DOI:  https://doi.org/10.1016/j.yexcr.2023.113715
Commun Biol. 2023 07 12. 6(1): 715

Centripetal nuclear shape fluctuations associate with chromatin condensation in early prophase.

Viola Introini, Gururaj Rao Kidiyoor, Giancarlo Porcella, Pietro Cicuta, Marco Cosentino Lagomarsino.

The nucleus plays a central role in several key cellular processes, including chromosome organisation, DNA replication and gene transcription. Recent work suggests an association between nuclear mechanics and cell-cycle progression, but many aspects of this connection remain unexplored. Here, by monitoring nuclear shape fluctuations at different cell cycle stages, we uncover increasing inward fluctuations in late G2 and in early prophase, which are initially transient, but develop into instabilities when approaching the nuclear-envelope breakdown. We demonstrate that such deformations correlate with chromatin condensation by perturbing both the chromatin and the cytoskeletal structures. We propose that the contrasting forces between an extensile stress and centripetal pulling from chromatin condensation could mechanically link chromosome condensation with nuclear-envelope breakdown, two main nuclear processes occurring during mitosis.

DOI: https://doi.org/10.1038/s42003-023-05074-9
iScience. 2023 Jul 21. 26(7): 107129

Polarized SCAR and the Arp2/3 complex regulate apical cortical remodeling in asymmetrically dividing neuroblasts.

Giulia Cazzagon, Chantal Roubinet, Buzz Baum.

  Although the formin-nucleated actomyosin cortex has been shown to drive the changes in cell shape that accompany animal cell division in both symmetric and asymmetric cell divisions, the mitotic role of cortical Arp2/3-nucleated actin networks remain unclear. Here using asymmetrically dividing Drosophila neural stem cells as a model system, we identify a pool of membrane protrusions that form at the apical cortex of neuroblasts as they enter mitosis. Strikingly, these apically localized protrusions are enriched in SCAR, and depend on SCAR and Arp2/3 complexes for their formation. Because compromising SCAR or the Arp2/3 complex delays the apical clearance of Myosin II at the onset of anaphase and induces cortical instability at cytokinesis, these data point to a role for an apical branched actin filament network in fine-tuning the actomyosin cortex to enable the precise control of cell shape changes during an asymmetric cell division.

Keywords:  Biological sciences; Cell biology; Molecular biology; Neuroscience

DOI:  https://doi.org/10.1016/j.isci.2023.107129
Dev Growth Differ. 2023 Jul 08.

The Crucial Role of CTCF in Mitotic Progression during Early Development of Sea Urchin.

Kaichi Watanabe, Megumi Fujita, Kazuko Okamoto, Hajime Yoshioka, Miki Moriwaki, Hideki Tagashira, Akinori Awazu, Takashi Yamamoto, Naoaki Sakamoto.

  CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. The expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula-to-blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development. This article is protected by copyright. All rights reserved.

Keywords:  CTCF; cell cycle; development; mitosis; sea urchin

DOI:  https://doi.org/10.1111/dgd.12875
Cancers (Basel). 2023 Jun 23. pii: 3309. [Epub ahead of print]15(13):

MCAK Inhibitors Induce Aneuploidy in Triple-Negative Breast Cancer Models.

John C Smith, Stefan Husted, Jay Pilrose, Stephanie C Ems-McClung, Jane R Stout, Richard L Carpenter, Claire E Walczak.

  Standard of care for triple-negative breast cancer (TNBC) involves the use of microtubule poisons such as paclitaxel, which are proposed to work by inducing lethal levels of aneuploidy in tumor cells. While these drugs are initially effective in treating cancer, dose-limiting peripheral neuropathies are common. Unfortunately, patients often relapse with drug-resistant tumors. Identifying agents against targets that limit aneuploidy may be a valuable approach for therapeutic development. One potential target is the microtubule depolymerizing kinesin, MCAK, which limits aneuploidy by regulating microtubule dynamics during mitosis. Using publicly available datasets, we found that MCAK is upregulated in triple-negative breast cancer and is associated with poorer prognoses. Knockdown of MCAK in tumor-derived cell lines caused a two- to five-fold reduction in the IC50 for paclitaxel, without affecting normal cells. Using FRET and image-based assays, we screened compounds from the ChemBridge 50 k library and discovered three putative MCAK inhibitors. These compounds reproduced the aneuploidy-inducing phenotype of MCAK loss, reduced clonogenic survival of TNBC cells regardless of taxane-resistance, and the most potent of the three, C4, sensitized TNBC cells to paclitaxel. Collectively, our work shows promise that MCAK may serve as both a biomarker of prognosis and as a therapeutic target.

Keywords:  MCAK/KIF2C; aneuploidy; chromosomal instability; drug discovery; triple-negative breast cancer

DOI:  https://doi.org/10.3390/cancers15133309