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ELife Dec 2022The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different...
The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different functional outcomes according to T-cell subsets. Whether the mitotic machineries supporting these qualitatively distinct proliferative responses are identical remains unknown. Here, we show that disruption of the microtubule-associated protein LIS1 in mouse models leads to proliferative defects associated with a blockade of T-cell development after β-selection and of peripheral CD4+ T-cell expansion after antigen priming. In contrast, cell divisions in CD8+ T cells occurred independently of LIS1 following T-cell antigen receptor stimulation, although LIS1 was required for proliferation elicited by pharmacological activation. In thymocytes and CD4+ T cells, LIS1 deficiency did not affect signaling events leading to activation but led to an interruption of proliferation after the initial round of division and to p53-induced cell death. Proliferative defects resulted from a mitotic failure, characterized by the presence of extra-centrosomes and the formation of multipolar spindles, causing abnormal chromosomes congression during metaphase and separation during telophase. LIS1 was required to stabilize dynein/dynactin complexes, which promote chromosome attachment to mitotic spindles and ensure centrosome integrity. Together, these results suggest that proliferative responses are supported by distinct mitotic machineries across T-cell subsets.
Topics: Animals; Mice; Cell Lineage; Centrosome; Chromosome Segregation; Dyneins; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus; T-Lymphocytes; 1-Alkyl-2-acetylglycerophosphocholine Esterase
PubMed: 36519536
DOI: 10.7554/eLife.80277 -
Communications Biology Dec 2022Faithful chromosome segregation requires bi-oriented kinetochore-microtubule attachment on the metaphase spindle. Aurora B kinase, the catalytic core of the chromosome...
Faithful chromosome segregation requires bi-oriented kinetochore-microtubule attachment on the metaphase spindle. Aurora B kinase, the catalytic core of the chromosome passage complex (CPC), plays a crucial role in this process. Aurora B activation has widely been investigated in the context of protein phosphorylation. Here, we report that Aurora B is ubiquitinated in mitosis through lysine-63 ubiquitin chains (K63-Ub), which is required for its activation. Mutation of Aurora B at its primary K63 ubiquitin site inhibits its activation, reduces its kinase activity, and disrupts the association of Aurora B with other components of CPC, leading to severe mitotic defects and cell apoptosis. Moreover, we identify that BRCC36 isopeptidase complex (BRISC) is the K63-specific deubiquitinating enzyme for Aurora B. BRISC deficiency augments the accumulation of Aurora B K63-Ubs, leading to Aurora B hyperactivation and erroneous chromosome-microtubule attachments. These findings define the role of K63-linked ubiquitination in regulating Aurora B activation and provide a potential site for Aurora B-targeting drug design.
Topics: Aurora Kinase B; Lysine; Ubiquitin; Deubiquitinating Enzymes
PubMed: 36473924
DOI: 10.1038/s42003-022-04299-4 -
Journal of Visualized Experiments : JoVE Nov 2022The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the...
The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.
Topics: Mice; Animals; Microtubule-Organizing Center; Spindle Apparatus; Sexual Maturation; Oocytes; Chromosome Segregation; Laser Therapy; Mammals
PubMed: 36440837
DOI: 10.3791/64439 -
Life Science Alliance Jan 2023Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also...
Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. Here, we show that acute disruption of the ER around spindle poles affects mitotic spindle size and function in syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. We reveal that when centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, we found that nuclear separation and distribution are affected by ER disruption. Our results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions.
Topics: Animals; Spindle Apparatus; Centrosome; Microtubules; Drosophila Proteins; Endoplasmic Reticulum; Drosophila
PubMed: 36379670
DOI: 10.26508/lsa.202201540 -
Molecular Biology of the Cell Jan 2023Faithful chromosome segregation requires the assembly of a bipolar spindle, consisting of two antiparallel microtubule (MT) arrays having most of their minus ends...
Faithful chromosome segregation requires the assembly of a bipolar spindle, consisting of two antiparallel microtubule (MT) arrays having most of their minus ends focused at the spindle poles and their plus ends overlapping in the spindle midzone. Spindle assembly, chromosome alignment, and segregation require highly dynamic MTs. The plus ends of MTs have been extensively investigated but their minus-end structure remains poorly characterized. Here, we used large-scale electron tomography to study the morphology of the MT minus ends in three dimensionally reconstructed metaphase spindles in HeLa cells. In contrast to the homogeneous open morphology of the MT plus ends at the kinetochores, we found that MT minus ends are heterogeneous, showing either open or closed morphologies. Silencing the minus end-specific stabilizer, MCRS1 increased the proportion of open MT minus ends. Altogether, these data suggest a correlation between the morphology and the dynamic state of the MT ends. Taking this heterogeneity of the MT minus-end morphologies into account, our work indicates an unsynchronized behavior of MTs at the spindle poles, thus laying the groundwork for further studies on the complexity of MT dynamics regulation.
Topics: Humans; HeLa Cells; Kinesins; Spindle Apparatus; Microtubules; Nuclear Proteins; RNA-Binding Proteins
PubMed: 36350698
DOI: 10.1091/mbc.E22-08-0306-T -
Nature Communications Nov 2022Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often...
Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic divisions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic division has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic division can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism.
Topics: Humans; Embryo, Mammalian; Chromosome Segregation; Mosaicism; Metaphase; Karyotype; Blastocyst; Aneuploidy
PubMed: 36347869
DOI: 10.1038/s41467-022-34294-6 -
Proceedings of the National Academy of... Nov 2022How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how...
How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.
Topics: Nuclear Matrix; Cytoskeleton; Microtubules; Mitosis; Extracellular Matrix; Spindle Apparatus; Anaphase
PubMed: 36322767
DOI: 10.1073/pnas.2116167119 -
Frontiers in Cell and Developmental... 2022Hybridogenesis is a hemiclonal reproductive strategy in diploid and triploid hybrids. Our study model is a frog (diploid RL and triploids RLL and RRL), a natural hybrid...
Hybridogenesis is a hemiclonal reproductive strategy in diploid and triploid hybrids. Our study model is a frog (diploid RL and triploids RLL and RRL), a natural hybrid between (LL) and (RR). Hybridogenesis relies on elimination of one genome (L or R) from gonocytes (G) in tadpole gonads during prespermatogenesis, but not from spermatogonial stem cells (SSCs) in adults. Here we provide the first comprehensive study of testis morphology combined with chromosome composition in the full spectrum of spermatogenic cells. Using genomic hybridization (GISH) and FISH we determined genomes in metaphase plates and interphase nuclei in Gs and SSCs. We traced genomic composition of SSCs, spermatocytes and spermatozoa in individual adult males that were crossed with females of the parental species and gave progeny. Degenerating gonocytes (24%-39%) and SSCs (18%-20%) led to partial sterility of juvenile and adult gonads. We conclude that elimination and endoreplication not properly completed during prespermatogenesis may be halted when gonocytes become dormant in juveniles. After resumption of mitotic divisions by SSCs in adults, these 20% of cells with successful genome elimination and endoreplication continue spermatogenesis, while in about 80% spermatogenesis is deficient. Majority of abnormal cells are eliminated by cell death, however some of them give rise to aneuploid spermatocytes and spermatozoa which shows that hybridogenesis is a wasteful process.
PubMed: 36313575
DOI: 10.3389/fcell.2022.1008506 -
Pharmaceuticals (Basel, Switzerland) Sep 2022Cancer continues to be one of the world's most severe public health issues. Polo-like kinase 1 (PLK1) is one of the most studied members of the polo-like kinase...
Cancer continues to be one of the world's most severe public health issues. Polo-like kinase 1 (PLK1) is one of the most studied members of the polo-like kinase subfamily of serine/threonine protein kinases. PLK1 is a key mitotic regulator responsible for cell cycle processes, such as mitosis initiation, bipolar mitotic spindle formation, centrosome maturation, the metaphase to anaphase transition, and mitotic exit, whose overexpression is often associated with oncogenesis. Moreover, it is also involved in DNA damage response, autophagy, cytokine signaling, and apoptosis. Due to its fundamental role in cell cycle regulation, PLK1 has been linked to various types of cancer onset and progression, such as lung, colon, prostate, ovary, breast cancer, melanoma, and AML. Hence, PLK1 is recognized as a critical therapeutic target in the treatment of various proliferative diseases. PLK1 inhibitors developed in recent years have been researched and studied through clinical trials; however, most of them have failed because of their toxicity and poor therapeutic response. To design more potent and selective PLK1 inhibitors, we performed a receptor-based hybrid 3D-QSAR study of two datasets, possessing similar common scaffolds. The developed hybrid CoMFA ( = 0.628, = 0.905) and CoMSIA ( = 0.580, = 0.895) models showed admissible statistical results. Comprehensive, molecular docking of one of the most active compounds from the dataset and hybrid 3D-QSAR studies revealed important active site residues of PLK1 and requisite structural characteristics of ligand to design potent PLK1 inhibitors. Based on this information, we have proposed approximately 38 PLK1 inhibitors. The newly designed PLK1 inhibitors showed higher activity (predicted pIC) than the most active compounds of all the derivatives selected for this study. We selected and synthesized two compounds, which were ultimately found to possess good IC values. Our design strategy provides insight into development of potent and selective PLK1 inhibitors.
PubMed: 36297281
DOI: 10.3390/ph15101170 -
The Journal of Cell Biology Jan 2023Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their...
Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar, and astral microtubules (kMTs, iMTs, and aMTs, respectively). Among all, aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, important for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/CCdc20). These data highlight a unique role for mitotic cyclin Clb4 in controlling aMT regulating factors, of which Kip2 is a prime candidate, provide a framework to understand aMT regulation in vertebrates, and uncover mechanistic principles of how the APC/CCdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.
Topics: Animals; Anaphase; Anaphase-Promoting Complex-Cyclosome; Cyclins; Microtubules; Spindle Apparatus; Cdc20 Proteins; Cyclin B
PubMed: 36269172
DOI: 10.1083/jcb.202203089