bims-micesi 2024-02-18 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–02–18
ten papers selected by
Valentina Piano, Uniklinik Köln

A conserved CENP-E region mediates BubR1-independent recruitment to the outer corona at mitotic onset.
Auxin-induced degradation of the aurora A kinase, AIR-1, in C. elegans does not prevent assembly of bipolar meiotic spindles.
Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation.
The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.
Efficient mitosis detection: leveraging pre-trained faster R-CNN and cell-level classification.
Search for chromosomal instability aiding variants reveal naturally occurring kinetochore gene variants that perturb chromosome segregation.
Generation of aneuploid cells and assessment of their ability to survive in presence of chemotherapeutic agents.
Switching from weak to strong cortical attachment of microtubules accounts for the transition from nuclear centration to spindle elongation in metazoans.
Specialized replication mechanisms maintain genome stability at human centromeres.
Loss of symmetric cell division of apical neural progenitors drives DENND5A -related developmental and epileptic encephalopathy.

Curr Biol. 2024 Feb 08. pii: S0960-9822(24)00076-9. [Epub ahead of print]

A conserved CENP-E region mediates BubR1-independent recruitment to the outer corona at mitotic onset.

Jeraldine Weber, Thibault Legal, Alicia Perez Lezcano, Agata Gluszek-Kustusz, Calum Paterson, Susana Eibes, Marin Barisic, Owen R Davies, Julie P I Welburn.

  The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091-2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression.

Keywords:  CENP-E motor; centromere; dynein; kinetochore; microtubule; mitosis; motor; outer corona; prometaphase; spindle assembly checkpoint

DOI:  https://doi.org/10.1016/j.cub.2024.01.042
MicroPubl Biol. 2024 ;2024

Auxin-induced degradation of the aurora A kinase, AIR-1, in C. elegans does not prevent assembly of bipolar meiotic spindles.

Karen McNally, Francis McNally.

Chromosome segregation during mitosis and male meiosis is mediated by centrosomal spindles that require the activity of the aurora A kinase, whereas female meiotic spindles of many species are acentrosomal. We addressed the role of the C. elegans aurora A kinase, AIR-1 , in acentrosomal spindle assembly by generating a strain in which AIR-1 is tagged with both an auxin-induced degron and HALO tag. The meiotic spindle pole marker, MEI-1 , and chromosomes were labeled with GFP and mCH::histone respectively. All meiotic spindles were bipolar in AIR-1 depleted embryos, however an increase in lagging chromosomes was observed during anaphase.

DOI: https://doi.org/10.17912/micropub.biology.001123
Methods Cell Biol. 2024 ;pii: S0091-679X(22)00173-X. [Epub ahead of print]182 1-20

Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation.

Yu-Fen Lin, Qing Hu, Alison Guyer, Daniele Fachinetti, Peter Ly.

  Chromothripsis describes the catastrophic fragmentation of individual chromosomes followed by its haphazard reassembly into a derivative chromosome harboring complex rearrangements. This process can be initiated by mitotic cell division errors when one or more chromosomes aberrantly mis-segregate into micronuclei and acquire extensive DNA damage. Approaches to induce the formation of micronuclei encapsulating random chromosomes have been used; however, the eventual reincorporation of the micronucleated chromosome into daughter cell nuclei poses a challenge in tracking the chromosome for multiple cell cycles. Here we outline an approach to genetically engineer stable human cell lines capable of efficient chromosome-specific micronuclei induction. This strategy, which targets the CENP-B-deficient Y chromosome centromere for inactivation, allows the stepwise process of chromothripsis to be experimentally recapitulated, including the mechanisms and timing of chromosome fragmentation. Lastly, we describe the integration of a selection marker onto the micronucleated Y chromosome that enables the diverse genomic rearrangement landscape arising from micronuclei formation to be interrogated.

Keywords:  Centromere; Chromosome segregation; Chromothripsis; Micronuclei

DOI:  https://doi.org/10.1016/bs.mcb.2022.10.009
Nat Commun. 2024 Feb 15. 15(1): 1385

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

Christopher B Nelson, Samuel Rogers, Kaushik Roychoudhury, Yaw Sing Tan, Caroline J Atkinson, Alexander P Sobinoff, Christopher G Tomlinson, Anton Hsu, Robert Lu, Eloise Dray, Michelle Haber, Jamie I Fletcher, Anthony J Cesare, Rashmi S Hegde, Hilda A Pickett.

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

DOI: https://doi.org/10.1038/s41467-024-45683-4
Biomed Phys Eng Express. 2024 Feb 15. 10(2):

Efficient mitosis detection: leveraging pre-trained faster R-CNN and cell-level classification.

Abdul R Shihabuddin, Sabeena Beevi K.

  The assessment of mitotic activity is an integral part of the comprehensive evaluation of breast cancer pathology. Understanding the level of tumor dissemination is essential for assessing the severity of the malignancy and guiding appropriate treatment strategies. A pathologist must manually perform the intricate and time-consuming task of counting mitoses by examining biopsy slices stained with Hematoxylin and Eosin (H&E) under a microscope. Mitotic cells can be challenging to distinguish in H&E-stained sections due to limited available datasets and similarities among mitotic and non-mitotic cells. Computer-assisted mitosis detection approaches have simplified the whole procedure by selecting, detecting, and labeling mitotic cells. Traditional detection strategies rely on image processing techniques that apply custom criteria to distinguish between different aspects of an image. Additionally, the automatic feature extraction from histopathology images that exhibit mitosis using neural networks.Additionally, the possibility of automatically extracting features from histopathological images using deep neural networks was investigated. This study examines mitosis detection as an object detection problem using multiple neural networks. From a medical standpoint, mitosis at the tissue level was also investigated utilising pre-trained Faster R-CNN and raw image data. Experiments were done on the MITOS-ATYPIA- 14 dataset and TUPAC16 dataset, and the results were compared to those of other methods described in the literature.

Keywords:  CNN; Faster R-CNN; MITOS-ATYPIA-14; mitosis; multiCNN

DOI:  https://doi.org/10.1088/2057-1976/ad262f
iScience. 2024 Mar 15. 27(3): 109007

Search for chromosomal instability aiding variants reveal naturally occurring kinetochore gene variants that perturb chromosome segregation.

Asifa Islam, Janeth Catalina Manjarrez-González, Xinhong Song, Trupti Gore, Viji M Draviam.

  Chromosomal instability (CIN) is a hallmark of cancers, and CIN-promoting mutations are not fully understood. Here, we report 141 chromosomal instability aiding variant (CIVa) candidates by assessing the prevalence of loss-of-function (LoF) variants in 135 chromosome segregation genes from over 150,000 humans. Unexpectedly, we observe both heterozygous and homozygous CIVa in Astrin and SKA3, two evolutionarily conserved kinetochore and microtubule-associated proteins essential for chromosome segregation. To stratify harmful versus harmless variants, we combine live-cell microscopy and controlled protein expression. We find the naturally occurring Astrin p.Q1012∗ variant is harmful as it fails to localize normally and induces chromosome misalignment and missegregation, in a dominant negative manner. In contrast, the Astrin p.L7Qfs∗21 variant generates a shorter isoform that localizes and functions normally, and the SKA3 p.Q70Kfs∗7 variant allows wild-type SKA complex localisation and function, revealing distinct resilience mechanisms that render these variants harmless. Thus, we present a scalable framework to predict and stratify naturally occurring CIVa, and provide insight into resilience mechanisms that compensate for naturally occurring CIVa.

Keywords:  Cell biology; Chromosome organization; Genotyping; Molecular genetics; Phenotyping; Techniques in genetics

DOI:  https://doi.org/10.1016/j.isci.2024.109007
Methods Cell Biol. 2024 ;pii: S0091-679X(22)00176-5. [Epub ahead of print]182 21-33

Generation of aneuploid cells and assessment of their ability to survive in presence of chemotherapeutic agents.

Marica Rosaria Ippolito, Stefano Santaguida.

  Aneuploidy is a condition in which cells have an abnormal number of chromosomes that is not a multiple of the haploid complement. It is known that aneuploidy has detrimental consequences on cell physiology, such as genome instability, metabolic and proteotoxic stress and decreased cellular fitness. Importantly, aneuploidy is a hallmark of tumors and it is associated with resistance to chemotherapeutic agents and poor clinical outcome. To shed light into how aneuploidy contributes to chemoresistance, we induced chromosome mis-segregation in human cancer cell lines, then treated them with several chemotherapeutic agents and evaluated the emergence of chemoresistance. By doing so, we found that elevation of chromosome mis-segregation promotes resistance to chemotherapeutic agents through the expansion of aneuploid karyotypes and subsequent selection of specific aneuploidies essential for cellular viability under those stressful conditions. Here, we describe a method to generate aneuploid cell populations and to evaluate their resistance to anti-cancer agents. This protocol has been already successfully employed and can be further utilized to accelerate the exploration of the role of aneuploidy in chemoresistance.

Keywords:  Aneuploidy; Chemoresistance; Genome instability; Mitotic errors; Mps1

DOI:  https://doi.org/10.1016/bs.mcb.2022.10.012
Heliyon. 2024 Feb 15. 10(3): e25494

Switching from weak to strong cortical attachment of microtubules accounts for the transition from nuclear centration to spindle elongation in metazoans.

Shohei Tada, Yoshitaka Yamazaki, Kazunori Yamamoto, Ken Fujii, Takahiro G Yamada, Noriko F Hiroi, Akatsuki Kimura, Akira Funahashi.

  The centrosome is a major microtubule organizing center in animal cells. The position of the centrosomes inside the cell is important for cell functions such as cell cycle, and thus should be tightly regulated. Theoretical models based on the forces generated along the microtubules have been proposed to account for the dynamic movements of the centrosomes during the cell cycle. These models, however, often adopted inconsistent assumptions to explain distinct but successive movements, thus preventing a unified model for centrosome positioning. For the centration of the centrosomes, weak attachment of the astral microtubules to the cell cortex was assumed. In contrast, for the separation of the centrosomes during spindle elongation, strong attachment was assumed. Here, we mathematically analyzed these processes at steady state and found that the different assumptions are proper for each process. We experimentally validated our conclusion using nematode and sea urchin embryos by manipulating their shapes. Our results suggest the existence of a molecular mechanism that converts the cortical attachment from weak to strong during the transition from centrosome centration to spindle elongation.

Keywords:  Astral microtubules; Cell cycle; Centrosome centration; Mathematical modeling; Spindle elongation

DOI:  https://doi.org/10.1016/j.heliyon.2024.e25494
Mol Cell. 2024 Feb 14. pii: S1097-2765(24)00054-6. [Epub ahead of print]

Specialized replication mechanisms maintain genome stability at human centromeres.

Andrea Scelfo, Annapaola Angrisani, Marco Grillo, Bethany M Barnes, Francesc Muyas, Carolin M Sauer, Chin Wei Brian Leung, Marie Dumont, Marine Grison, David Mazaud, Mickaël Garnier, Laetitia Guintini, Louisa Nelson, Fumiko Esashi, Isidro Cortés-Ciriano, Stephen S Taylor, Jérôme Déjardin, Therese Wilhelm, Daniele Fachinetti.

  The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes.

Keywords:  DNA damage; DNA replication; cancer; centromere; genome instability; mitosis; proteomics; recombination; replication stress

DOI:  https://doi.org/10.1016/j.molcel.2024.01.018
medRxiv. 2024 Jan 31. pii: 2022.08.23.22278845. [Epub ahead of print]

Loss of symmetric cell division of apical neural progenitors drives DENND5A -related developmental and epileptic encephalopathy.

Undiagnosed Diseases Network

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A -related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

DOI: https://doi.org/10.1101/2022.08.23.22278845