bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2024–06–16
twelve papers selected by
Valentina Piano, Uniklinik Köln
  1. Measuring and modeling the dynamics of mitotic error correction.
  2. Vertebrate centromere architecture: from chromatin threads to functional structures.
  3. Kinetoplastid kinetochore proteins KKT14-KKT15 are divergent Bub1/BubR1-Bub3 proteins.
  4. Torques within and outside the human spindle balance twist at anaphase.
  5. Molecular interplay between HURP and Kif18A in mitotic spindle regulation.
  6. Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis.
  7. Target Identification and Mechanistic Characterization of Indole Terpenoid Mimics: Proper Spindle Microtubule Assembly Is Essential for Cdh1-Mediated Proteolysis of CENP-A.
  8. A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development.
  9. Hyperactivation of p53 contributes to mitotic catastrophe in podocytes through regulation of the Wee1/CDK1/cyclin B1 axis.
  10. The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis.
  11. Transient PP2A SIP complex localization to mitotic SPBs for SIN inhibition is mediated solely by the Csc1 FHA domain.
  12. Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.
  1. Proc Natl Acad Sci U S A. 2024 Jun 18. 121(25): e2323009121
    Measuring and modeling the dynamics of mitotic error correction.
    Gloria Ha, Paul Dieterle, Hao Shen, Ariel Amir, Daniel J Needleman.
      Error correction is central to many biological systems and is critical for protein function and cell health. During mitosis, error correction is required for the faithful inheritance of genetic material. When functioning properly, the mitotic spindle segregates an equal number of chromosomes to daughter cells with high fidelity. Over the course of spindle assembly, many initially erroneous attachments between kinetochores and microtubules are fixed through the process of error correction. Despite the importance of chromosome segregation errors in cancer and other diseases, there is a lack of methods to characterize the dynamics of error correction and how it can go wrong. Here, we present an experimental method and analysis framework to quantify chromosome segregation error correction in human tissue culture cells with live cell confocal imaging, timed premature anaphase, and automated counting of kinetochores after cell division. We find that errors decrease exponentially over time during spindle assembly. A coarse-grained model, in which errors are corrected in a chromosome-autonomous manner at a constant rate, can quantitatively explain both the measured error correction dynamics and the distribution of anaphase onset times. We further validated our model using perturbations that destabilized microtubules and changed the initial configuration of chromosomal attachments. Taken together, this work provides a quantitative framework for understanding the dynamics of mitotic error correction.
    Keywords:  anaphase timing; chromosome segregation; coarse-grained modeling; error correction; mitosis
    DOI:  https://doi.org/10.1073/pnas.2323009121
  2. Chromosoma. 2024 Jun 10.
    Vertebrate centromere architecture: from chromatin threads to functional structures.
    Lorena Andrade Ruiz, Geert J P L Kops, Carlos Sacristan.
      Centromeres are chromatin structures specialized in sister chromatid cohesion, kinetochore assembly, and microtubule attachment during chromosome segregation. The regional centromere of vertebrates consists of long regions of highly repetitive sequences occupied by the Histone H3 variant CENP-A, and which are flanked by pericentromeres. The three-dimensional organization of centromeric chromatin is paramount for its functionality and its ability to withstand spindle forces. Alongside CENP-A, key contributors to the folding of this structure include components of the Constitutive Centromere-Associated Network (CCAN), the protein CENP-B, and condensin and cohesin complexes. Despite its importance, the intricate architecture of the regional centromere of vertebrates remains largely unknown. Recent advancements in long-read sequencing, super-resolution and cryo-electron microscopy, and chromosome conformation capture techniques have significantly improved our understanding of this structure at various levels, from the linear arrangement of centromeric sequences and their epigenetic landscape to their higher-order compaction. In this review, we discuss the latest insights on centromere organization and place them in the context of recent findings describing a bipartite higher-order organization of the centromere.
    Keywords:  CENP-A; Centromere; Chromatin organization; Epigenetics; Kinetochore
    DOI:  https://doi.org/10.1007/s00412-024-00823-z
  3. Open Biol. 2024 Jun;14(6): 240025
    Kinetoplastid kinetochore proteins KKT14-KKT15 are divergent Bub1/BubR1-Bub3 proteins.
    Daniel Ballmer, William Carter, Jolien J E van Hooff, Eelco C Tromer, Midori Ishii, Patryk Ludzia, Bungo Akiyoshi.
      Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.
    Keywords:  Trypanosoma brucei; chromosome segregation; kinetochore; kinetoplastid; spindle checkpoint
    DOI:  https://doi.org/10.1098/rsob.240025
  4. J Cell Biol. 2024 Sep 02. pii: e202312046. [Epub ahead of print]223(9):
    Torques within and outside the human spindle balance twist at anaphase.
    Lila Neahring, Nathan H Cho, Yifei He, 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, 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 together 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.
    DOI:  https://doi.org/10.1083/jcb.202312046
  5. Res Sq. 2024 May 29. pii: rs.3.rs-4249615. [Epub ahead of print]
    Molecular interplay between HURP and Kif18A in mitotic spindle regulation.
    Juan M Perez-Bertoldi, Yuanchang Zhao, Akanksha Thawani, Ahmet Yildiz, Eva Nogales.
      During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determined the binding mode of HURP to microtubules using Cryo-EM. The structure reveals that one HURP motif spans laterally across β-tubulin, while a second motif binds between adjacent protofilaments. HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observed that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in spindle length control.
    DOI:  https://doi.org/10.21203/rs.3.rs-4249615/v1
  6. bioRxiv. 2024 May 30. pii: 2024.05.29.596439. [Epub ahead of print]
    Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis.
    Andreas Brunner, Natalia Rosalia Morero, Wanlu Zhang, M Julius Hossain, Marko Lampe, Hannah Pflaumer, Aliaksandr Halavatyi, Jan-Michael Peters, Kai S Beckwith, Jan Ellenberg.
      How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early Gl, Cohesin-STAGl and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in Gl, is responsible for compaction inside TAD structures and forms paired complexes upon completed nuclear import. 0ur quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by sequential action of two Condensins in mitosis is seamlessly replaced by a less compact, but conceptually similar hierarchically nested loop architecture driven by sequential action of two Cohesins.
    DOI:  https://doi.org/10.1101/2024.05.29.596439
  7. Adv Sci (Weinh). 2024 Jun 14. e2305593
    Target Identification and Mechanistic Characterization of Indole Terpenoid Mimics: Proper Spindle Microtubule Assembly Is Essential for Cdh1-Mediated Proteolysis of CENP-A.
    Yan Peng, Yumeng Zhang, Ruan Fang, Hao Jiang, Gongcai Lan, Zhou Xu, Yajie Liu, Zhaoyang Nie, Lu Ren, Fengcan Wang, Shou-De Zhang, Yuyong Ma, Peng Yang, Hong-Hua Ge, Wei-Dong Zhang, Cheng Luo, Ang Li, Weiwei He.
      Centromere protein A (CENP-A), a histone H3 variant specific to centromeres, is crucial for kinetochore positioning and chromosome segregation. However, its regulatory mechanism in human cells remains incompletely understood. A structure-activity relationship (SAR) study of the cell-cycle-arresting indole terpenoid mimic JP18 leads to the discovery of two more potent analogs, (+)-6-Br-JP18 and (+)-6-Cl-JP18. Tubulin is identified as a potential cellular target of these halogenated analogs by using the drug affinity responsive target stability (DARTS) based method. X-ray crystallography analysis reveals that both molecules bind to the colchicine-binding site of β-tubulin. Treatment of human cells with microtubule-targeting agents (MTAs), including these two compounds, results in CENP-A accumulation by destabilizing Cdh1, a co-activator of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. This study establishes a link between microtubule dynamics and CENP-A accumulation using small-molecule tools and highlights the role of Cdh1 in CENP-A proteolysis.
    Keywords:  CENP‐A regulation; Cdh1; colchicine‐binding site inhibitor; indole terpenoid; target identification
    DOI:  https://doi.org/10.1002/advs.202305593
  8. Proc Natl Acad Sci U S A. 2024 Jun 18. 121(25): e2318838121
    A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development.
    Teije C Middelkoop, Jonas Neipel, Caitlin E Cornell, Ronald Naumann, Lokesh G Pimpale, Frank Jülicher, Stephan W Grill.
      Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.
    Keywords:  actomyosin; biophysics; cell biology; cytokinesis; development
    DOI:  https://doi.org/10.1073/pnas.2318838121
  9. Ren Fail. 2024 Dec;46(2): 2365408
    Hyperactivation of p53 contributes to mitotic catastrophe in podocytes through regulation of the Wee1/CDK1/cyclin B1 axis.
    Jie Feng, Liyi Xie, Wanhong Lu, Xiaoyang Yu, Hongjuan Dong, Yuefeng Ma, Ranran Kong.
      Podocyte loss in glomeruli is a fundamental event in the pathogenesis of chronic kidney diseases. Currently, mitotic catastrophe (MC) has emerged as the main cause of podocyte loss. However, the regulation of MC in podocytes has yet to be elucidated. The current work aimed to study the role and mechanism of p53 in regulating the MC of podocytes using adriamycin (ADR)-induced nephropathy. In vitro podocyte stimulation with ADR triggered the occurrence of MC, which was accompanied by hyperactivation of p53 and cyclin-dependent kinase (CDK1)/cyclin B1. The inhibition of p53 reversed ADR-evoked MC in podocytes and protected against podocyte injury and loss. Further investigation showed that p53 mediated the activation of CDK1/cyclin B1 by regulating the expression of Wee1. Restraining Wee1 abolished the regulatory effect of p53 inhibition on CDK1/cyclin B1 and rebooted MC in ADR-stimulated podocytes via p53 inhibition. In a mouse model of ADR nephropathy, the inhibition of p53 ameliorated proteinuria and podocyte injury. Moreover, the inhibition of p53 blocked the progression of MC in podocytes in ADR nephropathy mice through the regulation of the Wee1/CDK1/cyclin B1 axis. Our findings confirm that p53 contributes to MC in podocytes through regulation of the Wee1/CDK1/Cyclin B1 axis, which may represent a novel mechanism underlying podocyte injury and loss during the progression of chronic kidney disorder.
    Keywords:  CDK1; Wee1; cyclin B1; mitotic catastrophe; p53; podocyte
    DOI:  https://doi.org/10.1080/0886022X.2024.2365408
  10. EMBO Rep. 2024 Jun 14.
    The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis.
    Helena Bragulat-Teixidor, Keisuke Ishihara, Gréta Martina Szücs, Shotaro Otsuka.
      Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.
    Keywords:  Correlative Light-electron Microscopy; Endoplasmic Reticulum; Membrane Contact Site; Nuclear Envelope; Open Mitosis
    DOI:  https://doi.org/10.1038/s44319-024-00175-w
  11. Mol Biol Cell. 2024 Jun 12. mbcE24040196
    Transient PP2A SIP complex localization to mitotic SPBs for SIN inhibition is mediated solely by the Csc1 FHA domain.
    Alaina H Willet, Liping Ren, Lesley A Turner, Kathleen L Gould.
      Many organisms utilize an actin- and myosin-based cytokinetic ring to help complete cytokinesis. In Schizosaccharomyces pombe, the Septation Initiation Network (SIN) promotes proper CR function and stability. The SIN is a conserved and essential signaling network consisting of a GTPase and a cascade of kinases assembled at the spindle pole body (SPB). The PP2A SIN inhibitory phosphatase (SIP) complex related to the STRIPAK phosphatase complex is one inhibitor of SIN signaling. The SIP consists of Csc1, Csc2, Csc3, Csc4, Paa1, and the phosphatase subunit Ppa3. Here, we determine that the SIP is anchored at the SPB via the Csc1 FHA domain and that constitutive SPB localization of the SIP is lethal due to persistent SIN inhibition. Disrupting SIP docking at the SPB with a point mutation within the FHA domain or eliminating phosphatase activity by introducing a point mutation within Ppa3 resulted in intact SIP complexes without SIN inhibitory function. Lastly, we defined the unique features of Ppa3 that allow it, but not two other PP2A catalytic subunits, to incorporate into the SIP. Overall, we provide insight into how the SIP complex assembles, localizes, and functions to counteract the SIN with spatiotemporal precision during cytokinesis.
    DOI:  https://doi.org/10.1091/mbc.E24-04-0196
  12. Development. 2024 Jun 12. pii: dev.202577. [Epub ahead of print]
    Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.
    Tirthankar Ray, Damo Shi, Tony J C Harris.
      Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue, and compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa seems to form through tissue-autonomous generation of spindle-shaped cells which nematically align in response to confinement by surrounding tissue.
    Keywords:  Amnioserosa; Confinement; Drosophila embryo; Nematic order; Spindle-shape cells
    DOI:  https://doi.org/10.1242/dev.202577
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