bims-micesi 2023-03-26 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2023–03–26
eight papers selected by
Valentina Piano, Uniklinik Köln

Principles and dynamics of spindle assembly checkpoint signalling.
The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex.
Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by super-resolution microscopy.
Structural Reorganization and Relaxation Dynamics of Axially Stressed Chromosomes.
The cell cycle and cell size influence the rates of global cellular translation and transcription in fission yeast.
Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.
FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase.
Single-cell transcriptome analysis of male chicken germ cells reveals changes in signaling pathway-related gene expression profiles during mitotic arrest.

Nat Rev Mol Cell Biol. 2023 Mar 24.

Principles and dynamics of spindle assembly checkpoint signalling.

Andrew D McAinsh, Geert J P L Kops.

The transmission of a complete set of chromosomes to daughter cells during cell division is vital for development and tissue homeostasis. The spindle assembly checkpoint (SAC) ensures correct segregation by informing the cell cycle machinery of potential errors in the interactions of chromosomes with spindle microtubules prior to anaphase. To do so, the SAC monitors microtubule engagement by specialized structures known as kinetochores and integrates local mechanical and chemical cues such that it can signal in a sensitive, responsive and robust manner. In this Review, we discuss how SAC proteins interact to allow production of the mitotic checkpoint complex (MCC) that halts anaphase progression by inhibiting the anaphase-promoting complex/cyclosome (APC/C). We highlight recent advances aimed at understanding the dynamic signalling properties of the SAC and how it interprets various naturally occurring intermediate attachment states. Further, we discuss SAC signalling in the context of the mammalian multisite kinetochore and address the impact of the fibrous corona. We also identify current challenges in understanding how the SAC ensures high-fidelity chromosome segregation.

DOI: https://doi.org/10.1038/s41580-023-00593-z
Nat Commun. 2023 Mar 18. 14(1): 1529

The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex.

Chu Chen, Valentina Piano, Amal Alex, Simon J Y Han, Pim J Huis In 't Veld, Babhrubahan Roy, Daniel Fergle, Andrea Musacchio, Ajit P Joglekar.

The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.

DOI: https://doi.org/10.1038/s41467-023-37235-z
Mol Biol Cell. 2023 Mar 22. mbcE22080332

Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by super-resolution microscopy.

Elena Di Tommaso, Valeria de Turris, Pavan Choppakatla, Hironori Funabiki, Simona Giunta.

The human centromere comprises large arrays of repetitive alpha-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Since CENP-A is bound to only a fraction of the alpha-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of alpha-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the Centromere Chromosome Orientation Fluorescent In Situ Hybridization (Cen-CO-FISH) technique together with Structured Illumination Microscopy (SIM). Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that alpha-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in presence or absence of spindle's microtubules. Using expansion microscopy (ExM), we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of alpha-satellite DNA, often visible as a ring thicker at the outer surface oriented towards the kinetochore-microtubules interface. Collectively, our data provide a 3D reconstruction of alpha-satellite DNA along with CENP-A clusters that outline the overall architecture of the mitotic centromere. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E22-08-0332
Biophys J. 2023 Mar 22. pii: S0006-3495(23)00202-3. [Epub ahead of print]

Structural Reorganization and Relaxation Dynamics of Axially Stressed Chromosomes.

Benjamin S Ruben, Sumitabha Brahmachari, Vinícius G Contessoto, Ryan R Cheng, Antonio B Oliveira Junior, Michele Di Pierro, José N Onuchic.

Chromosomes endure mechanical stresses throughout the cell cycle, for example resulting from the pulling of chromosomes by spindle fibers during mitosis or deformation of the nucleus during cell migration. The response to physical stress is closely related to chromosome structure and function. Micromechanical studies of mitotic chromosomes have revealed them to be remarkably extensible objects and informed early models of mitotic chromosome organization. We use a data-driven, coarse-grained polymer modeling approach to explore the relationship between the spatial organization of individual chromosomes and their emergent mechanical properties. In particular, we investigate the mechanical properties of our model chromosomes by axially stretching them. Simulated stretching led to a linear force-extension curve for small strain, with mitotic chromosomes behaving about ten-fold stiffer than interphase chromosomes. Studying their relaxation dynamics, we found that chromosomes are viscoelastic solids with a highly liquid-like, viscous behavior in interphase that becomes solid-like in mitosis. This emergent mechanical stiffness originates from lengthwise compaction, an effective potential capturing the activity of loop-extruding SMC complexes. Chromosomes denature under large strains via unraveling, which is characterized by opening of large-scale folding patterns. By quantifying the effect of mechanical perturbations on the chromosome's structural features, our model provides a nuanced understanding of in vivo mechanics of chromosomes.

DOI: https://doi.org/10.1016/j.bpj.2023.03.029
EMBO J. 2023 Mar 23. e113333

The cell cycle and cell size influence the rates of global cellular translation and transcription in fission yeast.

Clovis Basier, Paul Nurse.

  How the production of biomass is controlled as cells increase in size and proceed through the cell cycle events is important for understanding the regulation of global cellular growth. This has been studied for decades but has not yielded consistent results, probably due to perturbations induced by the synchronisation methods used in most previous studies. To avoid this problem, we have developed a system to analyse unperturbed exponentially growing populations of fission yeast cells. We generated thousands of fixed single-cell measurements of cell size, cell cycle stage and the levels of global cellular translation and transcription. We show that translation scales with size, and additionally, increases at late S-phase/early G2 and early in mitosis and decreases later in mitosis, suggesting that cell cycle controls are also operative over global cellular translation. Transcription increases with both size and the amount of DNA, suggesting that the level of transcription of a cell may be the result of a dynamic equilibrium between the number of RNA polymerases associating and disassociating from DNA.

Keywords:  cell cycle; cell size; scaling; transcription; translation

DOI:  https://doi.org/10.15252/embj.2022113333
EMBO Rep. 2023 Mar 23. e56273

Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Jia-Qian Ju, Zhen-Nan Pan, Kun-Huan Zhang, Yi-Ming Ji, Jing-Cai Liu, Shao-Chen Sun.

  Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

Keywords:  G2/M transition; Mcrs1; meiosis; oocyte; spindle

DOI:  https://doi.org/10.15252/embr.202256273
bioRxiv. 2023 Mar 17. pii: 2023.03.06.531310. [Epub ahead of print]

FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase.

Jason S Wasserman, Bulat Faezov, Kishan R Patel, Alison N Kurimchak, Seren M Palacio, Holly Fowle, Brennan C McEwan, Qifang Xu, Ziran Zhao, Lauren Cressey, Neil Johnson, James S Duncan, Arminja N Kettenbach, Roland L Dunbrack, Xavier Graña.

The Ser/Thr protein phosphatase 2A (PP2A) is a highly conserved collection of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). PP2A/B55 substrate recruitment was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM [ RK ]- V -x-x-[ VI ]- R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A. This conserved SLiM is necessary for FAM122A binding to B55α in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational and biochemical data and supports a mechanism whereby FAM122A uses the 'SLiM' in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, progression through cell cycle transitions and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a 'SLiM'-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.

DOI: https://doi.org/10.1101/2023.03.06.531310
FEBS Open Bio. 2023 Mar 24.

Single-cell transcriptome analysis of male chicken germ cells reveals changes in signaling pathway-related gene expression profiles during mitotic arrest.

Hyeon Jeong Choi, Kyung Min Jung, Kyung Je Park, Kyung Youn Lee, Seung Je Woo, Jae Yong Han.

  Mitotic arrest is necessary for embryonic development of germ cells, and thus it is important to understand the signaling pathways that regulate mitotic arrest. Here, we investigated the signaling pathway dynamics of male embryonic chicken germ cells during mitotic arrest by single-cell transcriptome analysis using germ cell tracing models. We identified signaling pathways that change at the transcriptional level during chicken male germ cell development after sex determination. We found that several components of the BMP, Notch, and JAK-STAT signaling pathways were downregulated at the mitotic-arrest stage and were reactivated one week after hatching, when all germ cells are quiescent after entering mitotic arrest. In addition, the transcriptional levels of components of the MAPK, Hedgehog, and thyroid-hormone signaling pathways were steadily upregulated after mitotic arrest. This suggests cooperation of multiple signaling pathways during entry into mitotic arrest and subsequent quiescence of chicken male germ cells.

Keywords:  chicken; male germ cell; mitotic arrest; signaling pathway; single-cell RNA sequencing

DOI:  https://doi.org/10.1002/2211-5463.13600