bims-micesi 2023-09-03 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2023–09–03
ten papers selected by
Valentina Piano, Uniklinik Köln

CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly.
Cleavage furrow positioning in dividing Dictyostelium cells.
Genetic enhancers of partial PLK1 inhibition reveal hypersensitivity to kinetochore perturbations.
Maintaining transcriptional homeostasis during cell cycle.
Epigenetic inheritance and boundary maintenance at human centromeres.
Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex.
DNAJA2 deficiency activates cGAS-STING pathway via the induction of aberrant mitosis and chromosome instability.
Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.
Mutations in CCNB3 affect its location thus causing a multiplicity of phenotypes in human oocytes maturation by aberrant CDK1 activity and APC/C activity at different stages.
Quantification of Chromosomal Aberrations in Mammalian Cells.

Nat Commun. 2023 Sep 01. 14(1): 5317

CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly.

Susana Eibes, Girish Rajendraprasad, Claudia Guasch-Boldu, Mirela Kubat, Yulia Steblyanko, Marin Barisic.

Accurate chromosome segregation in mitosis depends on multiprotein structures called kinetochores that are built on the centromeric region of sister chromatids and serve to capture mitotic spindle microtubules. In early mitosis, unattached kinetochores expand a crescent-shaped structure called fibrous corona whose function is to facilitate initial kinetochore-microtubule attachments and chromosome transport by microtubules. Subsequently, the fibrous corona must be timely disassembled to prevent segregation errors. Although recent studies provided new insights on the molecular content and mechanism of fibrous corona assembly, it remains unknown what triggers the disassembly of the outermost and dynamic layer of the kinetochore. Here, we show that Aurora A and B kinases phosphorylate CENP-E to release it from an autoinhibited state. At kinetochores, Aurora B phosphorylates CENP-E to prevent its premature removal together with other corona proteins by dynein. At the spindle poles, Aurora A phosphorylates CENP-E to promote chromosome congression and prevent accumulation of corona proteins at the centrosomes, allowing for their intracellular redistribution. Thus, we propose the Aurora A/B-CENP-E axis as a critical element of the long-sought-for mechanism of fibrous corona disassembly that is essential for accurate chromosome segregation.

DOI: https://doi.org/10.1038/s41467-023-41091-2
Cytoskeleton (Hoboken). 2023 Aug 31.

Cleavage furrow positioning in dividing Dictyostelium cells.

Akiko Okada, Shigehiko Yumura.

  Accurate placement of the cleavage furrow is crucial for successful cell division. Recent advancements have revealed that diverse mechanisms have evolved across different branches of the phylogenetic tree. Here, we employed Dictyostelium cells to validate previous models. We observed that during metaphase and early anaphase, mitotic spindles exhibited random rotary movements which ceased when the spindle elongated by approximately 7 μm. At this point, astral microtubules reached the polar cell cortex and fixed the spindle axis, causing cells to elongate by extending polar pseudopods and divide along the spindle axis. Therefore, the position of the furrow is determined when the spindle orientation is fixed. The distal ends of astral microtubules stimulate the extension of pseudopods at the polar cortex. One signal for pseudopod extension may be phosphatidylinositol trisphosphate in the cell membrane, but there appears to be another unknown signal. At the onset of polar pseudopod extension, cortical flow began from both poles toward the equator. We suggest that polar stimulation by astral microtubules determines the furrow position, induces polar pseudopod extension and cortical flow, and accumulates the elements necessary for the construction of the contractile ring.

Keywords:  centrosome; cortical flow; cytokinesis; microtubule; mitotic spindle

DOI:  https://doi.org/10.1002/cm.21784
PLoS Genet. 2023 Aug 28. 19(8): e1010903

Genetic enhancers of partial PLK1 inhibition reveal hypersensitivity to kinetochore perturbations.

Karine Normandin, Jasmin Coulombe-Huntington, Corinne St-Denis, Alexandre Bernard, Mohammed Bourouh, Thierry Bertomeu, Mike Tyers, Vincent Archambault.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors.

DOI: https://doi.org/10.1371/journal.pgen.1010903
Transcription. 2023 Sep 01. 1-21

Maintaining transcriptional homeostasis during cell cycle.

Lucía Ramos-Alonso, Pierre Chymkowitch.

  The preservation of gene expression patterns that define cellular identity throughout the cell division cycle is essential to perpetuate cellular lineages. However, the progression of cells through different phases of the cell cycle severely disrupts chromatin accessibility, epigenetic marks, and the recruitment of transcriptional regulators. Notably, chromatin is transiently disassembled during S-phase and undergoes drastic condensation during mitosis, which is a significant challenge to the preservation of gene expression patterns between cell generations. This article delves into the specific gene expression and chromatin regulatory mechanisms that facilitate the preservation of transcriptional identity during replication and mitosis. Furthermore, we emphasize our recent findings revealing the unconventional role of yeast centromeres and mitotic chromosomes in maintaining transcriptional fidelity beyond mitosis.

Keywords:  Cell Fate; Cell cycle; centromere; chromatin; mitosis; replication; transcription memory

DOI:  https://doi.org/10.1080/21541264.2023.2246868
Curr Opin Struct Biol. 2023 Aug 30. pii: S0959-440X(23)00168-9. [Epub ahead of print]82 102694

Epigenetic inheritance and boundary maintenance at human centromeres.

Pragya Sidhwani, Aaron F Straight.

Centromeres are chromosomal regions that provide the foundation for microtubule attachment during chromosome segregation. Centromeres are epigenetically defined by nucleosomes containing the histone H3 variant centromere protein A (CENP-A) and, in many organisms, are surrounded by transcriptionally repressed pericentromeric chromatin marked by trimethylation of histone H3 lysine 9 (H3K9me3). Pericentromeric regions facilitate sister chromatid cohesion during mitosis, thereby supporting centromere function. Heterochromatin has a known propensity to spread into adjacent euchromatic domains unless it is properly bounded. Heterochromatin spreading into the centromere can disrupt kinetochore function, perturbing chromosome segregation and genome stability. In the fission yeast Schizosaccharomyces pombe, tRNA genes provide barriers to heterochromatin spread at the centromere, the absence of which results in abnormal meiotic chromosome segregation. How heterochromatin-centromere boundaries are established in humans is not understood. We propose models for stable epigenetic inheritance of centromeric domains in humans and discuss advances that will enable the discovery of novel regulators of this process.

DOI: https://doi.org/10.1016/j.sbi.2023.102694
Front Cell Dev Biol. 2023 ;11 1220529

Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex.

Amalia S Parra, Cameron A Moezzi, Christopher A Johnston.

  Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In Drosophila neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). Here we identify Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (HtsHEAD) to bind a short Mud coiled-coil domain (MudCC) that is adjacent to the Pins-binding domain (MudPBD). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. In vitro analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, we find that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via MudCC oligomerization) and protein disorder (via an intrinsically disordered region in Hts; HtsIDR) in phase separation of the Hts/Mud/Pins complex. Our study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output.

Keywords:  asymmetric cell division; cell polarity; neural stem cell; phase separation; spindle orientation

DOI:  https://doi.org/10.3389/fcell.2023.1220529
Nat Commun. 2023 Aug 28. 14(1): 5246

DNAJA2 deficiency activates cGAS-STING pathway via the induction of aberrant mitosis and chromosome instability.

Yaping Huang, Changzheng Lu, Hanzhi Wang, Liya Gu, Yang-Xin Fu, Guo-Min Li.

Molecular chaperone HSP70s are attractive targets for cancer therapy, but their substrate broadness and functional non-specificity have limited their role in therapeutical success. Functioning as HSP70's cochaperones, HSP40s determine the client specificity of HSP70s, and could be better targets for cancer therapy. Here we show that tumors defective in HSP40 member DNAJA2 are benefitted from immune-checkpoint blockade (ICB) therapy. Mechanistically, DNAJA2 maintains centrosome homeostasis by timely degrading key centriolar satellite proteins PCM1 and CEP290 via HSC70 chaperone-mediated autophagy (CMA). Tumor cells depleted of DNAJA2 or CMA factor LAMP2A exhibit elevated levels of centriolar satellite proteins, which causes aberrant mitosis characterized by abnormal spindles, chromosome missegregation and micronuclei formation. This activates the cGAS-STING pathway to enhance ICB therapy response in tumors derived from DNAJA2-deficient cells. Our study reveals a role for DNAJA2 to regulate mitotic division and chromosome stability and suggests DNAJA2 as a potential target to enhance cancer immunotherapy, thereby providing strategies to advance HSPs-based cancer therapy.

DOI: https://doi.org/10.1038/s41467-023-40952-0
Korean J Physiol Pharmacol. 2023 Sep 01. 27(5): 427-436

Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

Ju Hwan Kim, Rajnikant Patel.

  Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B), the human homologue of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares high sequence homology with Mad2, the mitotic checkpoint protein. Previously, we demonstrated the involvement of Mad2B in the cisplatin-induced DNA damage response. In this study, we extend our findings to show that Mad2B is recruited to sites of DNA damage in human cancer cells in response to cisplatin treatment. We found that in undamaged cells, Mad2B exists in a complex with Polζ-Rev1 and the APC/C subunit Cdc27. Following cisplatin-induced DNA damage, we observed an increase in the recruitment of Mad2B and Cdc20 (the activators of the APC/C), to the complex. The involvement of Mad2B-Cdc20-APC/C during DNA damage has not been reported before and suggests that the APC/C is activated following cisplatin-induced DNA damage. Using an in vitro ubiquitination assay, our data confirmed Mad2B-dependent activation of APC/C in cisplatin-treated cells. Mad2B may act as an accelerator for APC/C activation during DNA damage response. Our data strongly suggest a role for Mad2B-APC/C-Cdc20 in the ubiquitination of proteins involved in the DNA damage response.

Keywords:  Anaphase-promoting complex-cyclosome; Cell division cycle 20; DNA damage; DNA polymerase ζ; Mad2B

DOI:  https://doi.org/10.4196/kjpp.2023.27.5.427
J Ovarian Res. 2023 Aug 28. 16(1): 178

Mutations in CCNB3 affect its location thus causing a multiplicity of phenotypes in human oocytes maturation by aberrant CDK1 activity and APC/C activity at different stages.

Congjing Wang, Meng Xi Chen, Yuan Zhang, Xue Bai, Qiqi Cao, Jian Han, Nana Zhang, Chun Zhao, Xiufeng Ling, Ximan Rui, Yichun Guan, Junqiang Zhang, Ran Huo.

   BACKGROUND: Oocyte maturation arrest results in female infertility and the genetic etiology of this phenotype remains largely unknown. Previous studies have proven that cyclins play a significant role in the cell cycle both in meiosis and mitosis. Cyclin B3 (CCNB3) is one of the members of the cyclin family and its function in human oocyte maturation is poorly understood.
METHODS: 118 infertile patients were recruited and WES was performed for 68 independent females that experienced oocyte maturation arrest. Four mutations in CCNB3 were found and effects of these mutations were validated by Sanger sequencing and in vitro functional analyses.
RESULTS: We found these mutations altered the location of cyclin B3 which affected the function of cyclin dependent kinase 1 (CDK1) and led to mouse oocyte arrested at germinal vesicle (GV) stage. And then, low CDK1 activity influenced the degradation of cadherin 1 (CDH1) and the accumulation of cell division cycle 20 (CDC20) which are two types of anaphase-promoting complex/cyclosome (APC/C) activators and act in different stages of the cell cycle. Finally, APC/C activity was downregulated due to insufficient CDC20 level and resulted in oocyte metaphase I (MI) arrest. Moreover, we also found that the addition of PP1 inhibitor Okadic acid and CDK1 inhibitor Roscovitine at corresponding stages during oocyte in vitro maturation (IVM) significantly improved the maturation rates in CCNB3 mutant cRNAs injected oocytes. The above experiments were performed in mouse oocytes.
CONCLUSION: Here, we report five independent patients in which mutations in CCNB3 may be the cause of oocyte maturation arrest. Our findings shed lights on the critical role of CCNB3 in human oocyte maturation.

Keywords:  Anaphase-promoting complex/cyclosome; Cyclin B3; Female infertility; Oogenesis

DOI:  https://doi.org/10.1186/s13048-023-01229-8
Bio Protoc. 2023 Aug 20. 13(16): e4739

Quantification of Chromosomal Aberrations in Mammalian Cells.

Inés Paniagua, Jacqueline J L Jacobs.

  Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes orchestrating such resolution can lead to chromosomal instability, which appears as numerical and structural chromosome aberrations. Conventional cytogenetics remains as the golden standard method to detect naturally occurring chromosomal aberrations or those resulting from the treatment with genotoxic drugs. However, the success of cytogenetic studies depends on having high-quality chromosome spreads, which has been proven to be particularly challenging. Moreover, a lack of scoring guidelines and standardized methods for treating cells with genotoxic agents contribute to significant variability amongst different studies. Here, we report a simple and effective method for obtaining well-spread chromosomes from mammalian cells for the analysis of chromosomal aberrations. In this method, cells are (1) arrested in metaphase (when chromosome morphology is clearest), (2) swollen in hypotonic solution, (3) fixed before being dropped onto microscope slides, and (4) stained with DNA dyes to visualize the chromosomes. Metaphase chromosomes are then analyzed using high-resolution microscopy. We also provide examples, representative images, and useful guidelines to facilitate the scoring of the different chromosomal aberrations. This method can be used for the diagnosis of genetic diseases, as well as for cancer studies, by identifying chromosomal defects and providing insight into the cellular processes that influence chromosome integrity.

Keywords:  Cancer; Chromosomal instability; Congenital malignancies; DNA repair; DNA replication; Genome integrity; Mitosis

DOI:  https://doi.org/10.21769/BioProtoc.4739