bims-micesi 2022-10-16 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–10–16
eight papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation.
Inhibition of Mps1 kinase enhances taxanes efficacy in castration resistant prostate cancer.
A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1.
Microscopic Detection of DNA Synthesis in Early Mitosis at Repetitive lacO Sequences in Human Cells.
The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes.
Co-inhibition of Aurora A and Haspin kinases enhances survivin blockage and p53 induction for mitotic catastrophe and apoptosis in human colorectal cancer.
Molecular convergence by differential domain acquisition is a hallmark of chromosomal passenger complex evolution.
Arginylation Regulates Cytoskeleton Organization and Cell Division and Affects Mitochondria in Fission Yeast.

J Cell Biol. 2022 Dec 05. pii: e202205051. [Epub ahead of print]221(12):

Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation.

Margarida Dantas, Andreia Oliveira, Paulo Aguiar, Helder Maiato, Jorge G Ferreira.

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger cytoskeletal reorganization and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in cell-cycle regulation. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains unknown. Here, we identify a tension-dependent signal on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which unfolds the nucleus during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope and regulating the nuclear translocation of cyclin B1. Our data demonstrate how nuclear tension during the G2-M transition contributes to timely and efficient mitotic spindle assembly and prevents chromosomal instability.

DOI: https://doi.org/10.1083/jcb.202205051
Cell Death Dis. 2022 Oct 13. 13(10): 868

Inhibition of Mps1 kinase enhances taxanes efficacy in castration resistant prostate cancer.

Sadia Sarwar, Viacheslav M Morozov, Hamsa Purayil, Yehia Daaka, Alexander M Ishov.

Androgen ablation therapy is the standard of care for newly diagnosed prostate cancer (PC) patients. PC that relapsed after hormonal therapy, referred to as castration-resistant PC (CRPC), often presents with metastasis (mCRPC) and is the major cause of disease lethality. The few available therapies for mCRPC include the Taxanes Docetaxel (DTX) and Cabazitaxel (CBZ). Alas, clinical success of Taxanes in mCRPC is limited by high intrinsic and acquired resistance. Therefore, it remains essential to develop rationally designed treatments for managing therapy-resistant mCRPC disease. The major effect of Taxanes on microtubule hyper-polymerization is a prolonged mitotic block due to activation of the Spindle Assembly Checkpoint (SAC). Taxane-sensitive cells eventually inactivate SAC and exit mitosis by mitotic catastrophe, resulting in genome instability and blockade of proliferation. Resistant cells remain in mitotic block, and, upon drug decay, resume mitosis and proliferation, underlying one resistance mechanism. In our study we explored the possibility of forced mitotic exit to elevate Taxane efficacy. Inactivation of the SAC component, mitotic checkpoint kinase Mps1/TTK with a small molecule inhibitor (Msp1i), potentiated efficacy of Taxanes treatment in both 2D cell culture and 3D prostasphere settings. Mechanistically, Mps1 inhibition forced mitotic catastrophe in cells blocked in mitosis by Taxanes. Androgen receptor (AR), the main driver of PC, is often mutated or truncated in mCRPC. Remarkably, Mps1i significantly potentiated CBZ cytotoxicity regardless of AR status, in both AR-WT and in AR-truncated CRPC cells. Overall, our data demonstrate that forced mitotic exit by Mps1 inhibition potentiates Taxanes efficacy. Given that several Mps1i's are currently in different stages of clinical trials, our results point to Mps1 as a new therapeutic target to potentiate efficacy of Taxanes in mCRPC patients.

DOI: https://doi.org/10.1038/s41419-022-05312-8
Cell Rep. 2022 Oct 11. pii: S2211-1247(22)01325-0. [Epub ahead of print]41(2): 111475

A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1.

Lisa Donker, Ronja Houtekamer, Marjolein Vliem, François Sipieter, Helena Canever, Manuel Gómez-González, Miquel Bosch-Padrós, Willem-Jan Pannekoek, Xavier Trepat, Nicolas Borghi, Martijn Gloerich.

  Epithelial cell divisions are coordinated with cell loss to preserve epithelial integrity. However, how epithelia adapt their rate of cell division to changes in cell number, for instance during homeostatic turnover or wounding, is not well understood. Here, we show that epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell-cycle progression. As local cell density increases, tensile forces on E-cadherin adhesions are reduced, which prompts the accumulation of the G2 checkpoint kinase Wee1 and downstream inhibitory phosphorylation of Cdk1. Consequently, dense epithelia contain a pool of cells that are temporarily halted in G2 phase. These cells are readily triggered to divide following epithelial wounding due to the consequent increase in intercellular forces and resulting degradation of Wee1. Our data collectively show that epithelial cell division is controlled by a mechanical G2 checkpoint, which is regulated by cell-density-dependent intercellular forces sensed and transduced by E-cadherin adhesions.

Keywords:  CP: Cell biology; E-cadherin; G2 checkpoint; adherens junction; cell cycle; cell division; epithelial homeostasis; mechanical forces; mechanotransduction; mitosis

DOI:  https://doi.org/10.1016/j.celrep.2022.111475
Bio Protoc. 2022 Sep 05. pii: e4504. [Epub ahead of print]12(17):

Microscopic Detection of DNA Synthesis in Early Mitosis at Repetitive lacO Sequences in Human Cells.

Kazumasa Yoshida, Riko Ishimoto, Masatoshi Fujita.

  In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.

Keywords:   BrdU ; DNA repair ; DNA replication ; Immunofluorescence ; MiDAS ; Mitosis ; Repetitive DNA ; Replication stress

DOI:  https://doi.org/10.21769/BioProtoc.4504
Int J Mol Sci. 2022 Sep 27. pii: 11385. [Epub ahead of print]23(19):

The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes.

Tommy Stormberg, Yuri L Lyubchenko.

  CENP-A is a histone variant found in high abundance at the centromere in humans. At the centromere, this histone variant replaces the histone H3 found throughout the bulk chromatin. Additionally, the centromere comprises tandem repeats of α-satellite DNA, which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here, we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on α-satellite DNA did not show any positional preference along the substrate, which is similar to both H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on α-satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly.

Keywords:  CENP-A nucleosomes; alpha satellite DNA; atomic force microscopy; centromere chromatin; nanoscale structure of nucleosomes

DOI:  https://doi.org/10.3390/ijms231911385
Biochem Pharmacol. 2022 Oct 11. pii: S0006-2952(22)00383-5. [Epub ahead of print] 115289

Co-inhibition of Aurora A and Haspin kinases enhances survivin blockage and p53 induction for mitotic catastrophe and apoptosis in human colorectal cancer.

Chien-I Lin, Zan-Chu Chen, Chien-Hung Chen, Yun-Hsuan Chang, Tsai-Chia Lee, Tsai-Tai Tang, Tzu-Wei Yu, Chih-Man Yang, Ming-Chang Tsai, Chi-Chou Huang, Tzu-Wei Yang, Chun-Che Lin, Rou-Hsin Wang, Guang-Yuh Chiou, Yuh-Jyh Jong, Jui-I Chao.

  Colorectal cancer (CRC) is a leading cause and mortality worldwide. Aurora A and haspin kinases act pivotal roles in mitotic progression. However, the blockage of Aurora A and Haspin for CRC therapy is still unclear. Here we show that the Haspin and p-H3T3 protein levels were highly expressed in CRC tumor tissues of clinical patients. Overexpression of Haspin increased the protein levels of p-H3T3 and survivin in human CRC cells; conversely, the protein levels of p-H3T3 and survivin were decreased by the Haspin gene knockdown. Moreover, the gene knockdown of Aurora A induced abnormal chromosome segregation, mitotic catastrophe, and cell growth inhibition. Combined targeted by co-treatment of CHR6494, a Haspin inhibitor, and MLN8237, an Aurora A inhibitor, enhanced apoptosis and CRC tumor inhibition. MLN8237 and CHR6494 induced abnormal chromosome segregation and mitotic catastrophe. Meanwhile, MLN8237 and CHR6494 inhibited survivin protein levels but conversely induced p53 protein expression. Ectopic survivin expression by transfection with a survivin-expressed vector resisted the cell death in the MLN8237- and CHR6494-treated cells. In contrast, the existence of functional p53 increased the apoptotic levels by treatment with MLN8237 and CHR6494. Co-treatment of CHR6494 and MLN8237 enhanced the blockage of human CRC xenograft tumors in nude mice. Taken together, co-inhibition of Aurora A and Haspin enhances survivin inhibition, p53 pathway induction, mitotic catastrophe, apoptosis and tumor inhibition that may provide a potential strategy for CRC therapy.

Keywords:  Aurora A; Colorectal cancer; Haspin; Survivin; p53

DOI:  https://doi.org/10.1016/j.bcp.2022.115289
Proc Natl Acad Sci U S A. 2022 Oct 18. 119(42): e2200108119

Molecular convergence by differential domain acquisition is a hallmark of chromosomal passenger complex evolution.

Shinichiro Komaki, Eelco C Tromer, Geert De Jaeger, Nancy De Winne, Maren Heese, Arp Schnittger.

  The chromosomal passenger complex (CPC) is a heterotetrameric regulator of eukaryotic cell division, consisting of an Aurora-type kinase and a scaffold built of INCENP, Borealin, and Survivin. While most CPC components are conserved across eukaryotes, orthologs of the chromatin reader Survivin have previously only been found in animals and fungi, raising the question of how its essential role is carried out in other eukaryotes. By characterizing proteins that bind to the Arabidopsis Borealin ortholog, we identified BOREALIN RELATED INTERACTOR 1 and 2 (BORI1 and BORI2) as redundant Survivin-like proteins in the context of the CPC in plants. Loss of BORI function is lethal and a reduced expression of BORIs causes severe developmental defects. Similar to Survivin, we find that the BORIs bind to phosphorylated histone H3, relevant for correct CPC association with chromatin. However, this interaction is not mediated by a BIR domain as in previously recognized Survivin orthologs but by an FHA domain, a widely conserved phosphate-binding module. We find that the unifying criterion of Survivin-type proteins is a helix that facilitates complex formation with the other two scaffold components and that the addition of a phosphate-binding domain, necessary for concentration at the inner centromere, evolved in parallel in different eukaryotic groups. Using sensitive similarity searches, we find conservation of this helical domain between animals and plants and identify the missing CPC component in most eukaryotic supergroups. Interestingly, we also detect Survivin orthologs without a defined phosphate-binding domain, likely reflecting the situation in the last eukaryotic common ancestor.

Keywords:  cell division; evolution; microtuble cytoskeleton

DOI:  https://doi.org/10.1073/pnas.2200108119
Mol Cell Biol. 2022 Oct 13. e0026122

Arginylation Regulates Cytoskeleton Organization and Cell Division and Affects Mitochondria in Fission Yeast.

Li Chen, Anna Kashina.

  Protein arginylation mediated by arginyltransferase Ate1 is a posttranslational modification of emerging importance implicated in the regulation of mammalian embryogenesis, the cardiovascular system, tissue morphogenesis, cell migration, neurodegeneration, cancer, and aging. Ate1 deletion results in embryonic lethality in mice but does not affect yeast viability, making yeast an ideal system to study the molecular pathways regulated by arginylation. Here, we conducted a global analysis of cytoskeleton-related arginylation-dependent phenotypes in Schizosaccharomyces pombe, a fission yeast species that shares many fundamental features of higher eukaryotic cells. Our studies revealed roles of Ate1 in cell division, cell polarization, organelle transport, and interphase cytoskeleton organization and dynamics. We also found a role of Ate1 in mitochondria morphology and maintenance. Furthermore, targeted mass spectrometry analysis of the total Sc. pombe arginylome identified a number of arginylated proteins, including those that play direct roles in these processes; lack of their arginylation may be responsible for ate1-knockout phenotypes. Our work outlines global biological processes potentially regulated by arginylation and paves the way to unraveling the functions of protein arginylation that are conserved at multiple levels of evolution and potentially constitute the primary role of this modification in vivo.

Keywords:  actin; arginylation; cytoskeleton; fission yeast; mitosis

DOI:  https://doi.org/10.1128/mcb.00261-22