bims-micesi 2024-03-03 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–03–03
nine papers selected by
Valentina Piano, Uniklinik Köln

PLK1 phosphorylation of ZW10 guides accurate chromosome segregation in mitosis.
Male meiotic spindle poles are stabilized by TACC3 and cKAP5/chTOG differently from female meiotic or somatic mitotic spindles in mice.
CKAP5 stabilizes CENP-E at kinetochores by regulating microtubule-chromosome attachments.
Skp2-Cyclin A Interaction Is Necessary For Mitotic Entry and Maintenance of Diploidy.
Torques within and outside the human spindle balance twist at anaphase.
Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.
KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.
Control of meiotic entry by dual inhibition of a key mitotic transcription factor.
NEK2 plays an essential role in porcine embryonic development by maintaining mitotic division and DNA damage response via the Wnt/β-catenin signalling pathway.

J Mol Cell Biol. 2024 Feb 24. pii: mjae008. [Epub ahead of print]

PLK1 phosphorylation of ZW10 guides accurate chromosome segregation in mitosis.

Sm Faysal Bellah, Fangyuan Xiong, Zhen Dou, Fengrui Yang, Xing Liu, Xuebiao Yao, Xinjiao Gao, Liangyu Zhang.

  Stable transmission of genetic information during cell division requires faithful chromosome segregation. Mounting evidence has demonstrated that PLK1 dynamics at kinetochores control correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive. Here, we identified a regulatory mechanism by which PLK1-elicited ZW10 phosphorylation regulates spindle checkpoint silencing in mitosis. ZW10 is a cognate substrate of PLK1, and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10-Zwint1 interactions. Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes, while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase, in which sister chromatids entangled as cells entered anaphase. These findings reveal the previously uncharacterized PLK1-ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis.

Keywords:  PLK1; ZW10; kinetochore; mitosis; phosphorylation

DOI:  https://doi.org/10.1093/jmcb/mjae008
Sci Rep. 2024 02 27. 14(1): 4808

Male meiotic spindle poles are stabilized by TACC3 and cKAP5/chTOG differently from female meiotic or somatic mitotic spindles in mice.

Calvin Simerly, Emily Robertson, Caleb Harrison, Sydney Ward, Charlize George, Jasmine Deleon, Carrie Hartnett, Gerald Schatten.

Transforming acidic acid coiled-coil protein 3 (TACC3) and cytoskeleton associated protein 5 (cKAP5; or colonic hepatic tumor overexpressed gene, chTOG) are vital for spindle assembly and stabilization initiated through TACC3 Aurora-A kinase interaction. Here, TACC3 and cKAP5/chTOG localization with monospecific antibodies is investigated in eGFP-centrin-2- expressing mouse meiotic spermatocytes. Both proteins bind spermatocyte spindle poles but neither kinetochore nor interpolar microtubules, unlike in mitotic mouse fibroblasts or female meiotic oocyte spindles. Spermatocytes do not display a liquid-like spindle domain (LISD), although fusing them into maturing oocytes generates LISD-like TACC3 condensates around sperm chromatin but sparse microtubule assembly. Microtubule inhibitors do not reduce TACC3 and cKAP5/chTOG spindle pole binding. MLN 8237 Aurora-A kinase inhibitor removes TACC3, not cKAP5/chTOG, disrupting spindle organization, chromosome alignment, and impacting spindle pole γ-tubulin intensity. The LISD disruptor 1,6-hexanediol abolished TACC3 in spermatocytes, impacting spindle bipolarity and chromosome organization. Cold microtubule disassembly and rescue experiments in the presence of 1,6-hexanediol reinforce the concept that spermatocyte TACC3 spindle pole presence is not required for spindle pole microtubule assembly. Collectively, meiotic spermatocytes without a LISD localize TACC3 and cKAP5/chTOG exclusively at spindle poles to support meiotic spindle pole stabilization during male meiosis, different from either female meiosis or mitosis.

DOI: https://doi.org/10.1038/s41598-024-55376-z
EMBO Rep. 2024 Feb 29.

CKAP5 stabilizes CENP-E at kinetochores by regulating microtubule-chromosome attachments.

R Bhagya Lakshmi, Pinaki Nayak, Linoy Raz, Apurba Sarkar, Akshay Saroha, Pratibha Kumari, Vishnu M Nair, Delvin P Kombarakkaran, S Sajana, Sanusha M G, Sarit S Agasti, Raja Paul, Uri Ben-David, Tapas K Manna.

  Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.

Keywords:  CENP-E; CKAP5; Kinetochore; Microtubule; PP1

DOI:  https://doi.org/10.1038/s44319-024-00106-9
J Mol Biol. 2024 Feb 27. pii: S0022-2836(24)00083-4. [Epub ahead of print] 168505

Skp2-Cyclin A Interaction Is Necessary For Mitotic Entry and Maintenance of Diploidy.

Biju Vasavan, Nilanjana Das, Paria Kahnamouei, Chantelle Trombley, Andrew Swan.

  Skp2, the substrate recognition component of the SCFSkp2 ubiquitin ligase, has been implicated in the targeted destruction of a number of key cell cycle regulators and the promotion of S-phase. One of its critical targets is the Cyclin dependent kinase (Cdk) inhibitor p27, and indeed the overexpression of Skp2 in a number of cancers is directly correlated with the premature degradation of p27. Skp2 was first identified as a protein that interacts with Cyclin A in transformed cells, but its role in this complex has remained unclear. In this paper, we demonstrate that Skp2 interacts with Cyclin A in Drosophila and is required to maintain Cyclin A levels and permit mitotic entry. Failure of mitotic entry in Skp2 mutant cells results in polyploidy. If these cells enter mitosis again they are unable to properly segregate their chromosomes, leading to checkpoint dependent cell cycle arrest or apoptosis. Thus Skp2 is required for mitosis and for maintaining diploidy and genome stability.

Keywords:  Drosophila; Skp2; cell cycle; cyclin; mitosis

DOI:  https://doi.org/10.1016/j.jmb.2024.168505
bioRxiv. 2023 Dec 10. pii: 2023.12.10.570990. [Epub ahead of print]

Torques within and outside the human spindle balance twist at anaphase.

Lila Neahring, Yifei He, Nathan H Cho, Gaoxiang Liu, Jonathan Fernandes, Caleb J Rux, Konstantinos Nakos, Radhika Subramanian, Srigokul Upadhyayula, Ahmet Yildiz, Sophie Dumont.

  At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, using lattice light sheet microscopy, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are redundantly required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.

Keywords:  chirality; cortex; dynein; lattice light sheet microscopy; mitosis; motors; spindle; torque; twist

DOI:  https://doi.org/10.1101/2023.12.10.570990
EMBO J. 2024 Feb 29.

Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.

Laura Meißner, Lukas Niese, Irene Schüring, Aniruddha Mitra, Stefan Diez.

  During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed helical motion of anti-parallel microtubules around each other. The sidestepping ratio increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbited without forward motion, with nearly full motor extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, contributes to flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.

Keywords:  Helical Motion; Kinesin; Spindle; Torque

DOI:  https://doi.org/10.1038/s44318-024-00048-x
Chromosome Res. 2024 Feb 26. 32(1): 3

KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.

Ludmila Oliveira, Pavel Neumann, Yennifer Mata-Sucre, Yi-Tzu Kuo, André Marques, Veit Schubert, Jiří Macas.

  Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

Keywords:  CENH3; Centromere; KNL1; NDC80; immunolabeling; kinetochore

DOI:  https://doi.org/10.1007/s10577-024-09747-x
Elife. 2024 Feb 27. pii: RP90425. [Epub ahead of print]12

Control of meiotic entry by dual inhibition of a key mitotic transcription factor.

Elçin Ünal, Amanda J Su, Siri C Yendluri.

  The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a functional homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

Keywords:  LUTI; S. cerevisiae; cell cycle; chromosomes; gene expression; meiosis; mitosis; transcription factor; uORF

DOI:  https://doi.org/10.7554/eLife.90425
Cell Prolif. 2024 Mar 01. e13626

NEK2 plays an essential role in porcine embryonic development by maintaining mitotic division and DNA damage response via the Wnt/β-catenin signalling pathway.

Se-Been Jeon, Pil-Soo Jeong, Hyo-Gu Kang, Min Ju Kim, Ji Hyeon Yun, Kyung Seob Lim, Bong-Seok Song, Sun-Uk Kim, Seong-Keun Cho, Bo-Woong Sim.

NIMA-related kinase 2 (NEK2) is a serine/threonine protein kinase that regulates mitosis and plays pivotal roles in cell cycle regulation and DNA damage repair. However, its function in porcine embryonic development is unknown. In this study, we used an NEK2-specific inhibitor, JH295 (JH), to investigate the role of NEK2 in embryonic development and the underlying regulatory mechanisms. Inhibition of NEK2 after parthenogenesis activation or in vitro fertilization significantly reduced the rates of cleavage and blastocyst formation, the numbers of trophectoderm and total cells and the cellular survival rate compared with the control condition. NEK2 inhibition delayed cell cycle progression at all stages from interphase to cytokinesis during the first mitotic division; it caused abnormal nuclear morphology in two- and four-cell stage embryos. Additionally, NEK2 inhibition significantly increased DNA damage and apoptosis, and it altered the expression levels of DNA damage repair- and apoptosis-related genes. Intriguingly, NEK2 inhibition downregulated the expression of β-catenin and its downstream target genes. To validate the relationship between Wnt/β-catenin signalling and NEK2 during porcine embryonic development, we cultured porcine embryos in JH-treated medium with or without CHIR99021, a Wnt activator. CHIR99021 co-treatment strongly restored the developmental parameters reduced by NEK2 inhibition to control levels. Our findings suggest that NEK2 plays an essential role in porcine embryonic development by regulating DNA damage repair and normal mitotic division via the Wnt/β-catenin signalling pathway.

DOI: https://doi.org/10.1111/cpr.13626