bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2023–05–28
eight papers selected by
Valentina Piano, Uniklinik Köln
  1. Defining a core configuration for human centromeres during mitosis.
  2. An intrinsically disordered kinetochore protein coordinates mechanical regulation of chromosome segregation by dynein.
  3. An Aurora B-RPA signaling axis secures chromosome segregation fidelity.
  4. Mitotic bookmarking by SWI/SNF subunits.
  5. Large-scale phosphomimetic screening identifies phospho-modulated motif-based protein interactions.
  6. The structural basis of the multi-step allosteric activation of Aurora B kinase.
  7. Measurement of Microtubule Stability in Mammalian Cells.
  8. Cockayne syndrome group A protein localizes at centrosomes during mitosis and regulates Cyclin B1 ubiquitination.
  1. bioRxiv. 2023 May 11. pii: 2023.05.10.539634. [Epub ahead of print]
    Defining a core configuration for human centromeres during mitosis.
    Ayantika Sen Gupta, Chris Seidel, Dai Tsuchiya, Sean McKinney, Zulin Yu, Sarah Smith, Jay Unruh, Jennifer L Gerton.
      The biorientation of sister chromatids on the mitotic spindle, essential for accurate sister chromatid segregation, relies on critical centromere components including cohesin, the centromere-specific H3 variant CENP-A, and centromeric DNA. Centromeric DNA is highly variable between chromosomes yet must accomplish a similar function. Moreover, how the 50 nm cohesin ring, proposed to encircle sister chromatids, accommodates inter-sister centromeric distances of hundreds of nanometers on the metaphase spindle is a conundrum. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We used ChIP-seq and super-resolution microscopy to examine the geometry of essential centromeric components on human chromosomes. ChIP-seq demonstrates that cohesin subunits are depleted in α-satellite arrays where CENP-A nucleosomes and kinetochores assemble. Cohesin is instead enriched at pericentromeric DNA. Structured illumination microscopy of sister centromeres is consistent, revealing a non-overlapping pattern of CENP-A and cohesin. We used single particle averaging of hundreds of mitotic sister chromatids to develop an average centromere model. CENP-A clusters on sister chromatids, connected by α-satellite, are separated by ∼562 nm with a perpendicular intervening ∼190 nM wide axis of cohesin. Two differently sized α-satellite arrays on chromosome 7 display similar inter-sister CENP-A cluster distance, demonstrating different sized arrays can achieve a common spacing. Our data suggest a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes at the outer edge of extensible α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation for future studies of additional components required for centromere function.
    DOI:  https://doi.org/10.1101/2023.05.10.539634
  2. bioRxiv. 2023 May 09. pii: 2023.05.07.539709. [Epub ahead of print]
    An intrinsically disordered kinetochore protein coordinates mechanical regulation of chromosome segregation by dynein.
    Jessica M McGory, Dylan M Barcelos, Vikash Verma, Thomas J Maresca.
      Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation through a poorly understood pathway. Here we identify a physical linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ mediate the connection of Spc105 to dynein. Furthermore, a minimal segment of Spc105 that contains regions with a propensity to multimerize and binding motifs for Bub1 and BubR1 is sufficient to functionally link Spc105 to RZZ and dynein. Deletion of the minimal region from Spc105 reduces recruitment of its binding partners to bioriented kinetochores and causes chromosome mis-segregation. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and higher-order oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 is required to recruit a core pool of RZZ that modulates microtubule attachment stability to promote accurate chromosome segregation.
    DOI:  https://doi.org/10.1101/2023.05.07.539709
  3. Nat Commun. 2023 May 25. 14(1): 3008
    An Aurora B-RPA signaling axis secures chromosome segregation fidelity.
    Poonam Roshan, Sahiti Kuppa, Jenna R Mattice, Vikas Kaushik, Rahul Chadda, Nilisha Pokhrel, Brunda R Tumala, Aparna Biswas, Brian Bothner, Edwin Antony, Sofia Origanti.
      Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.
    DOI:  https://doi.org/10.1038/s41467-023-38711-2
  4. Nature. 2023 May 24.
    Mitotic bookmarking by SWI/SNF subunits.
    Zhexin Zhu, Xiaolong Chen, Ao Guo, Trishabelle Manzano, Patrick J Walsh, Kendall M Wills, Rebecca Halliburton, Sandi Radko-Juettner, Raymond D Carter, Janet F Partridge, Douglas R Green, Jinghui Zhang, Charles W M Roberts.
      For cells to initiate and sustain a differentiated state, it is necessary that a 'memory' of this state is transmitted through mitosis to the daughter cells1-3. Mammalian switch/sucrose non-fermentable (SWI/SNF) complexes (also known as Brg1/Brg-associated factors, or BAF) control cell identity by modulating chromatin architecture to regulate gene expression4-7, but whether they participate in cell fate memory is unclear. Here we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but are bound to promoters during mitosis, and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 has a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.
    DOI:  https://doi.org/10.1038/s41586-023-06085-6
  5. Mol Syst Biol. 2023 May 23. e11164
    Large-scale phosphomimetic screening identifies phospho-modulated motif-based protein interactions.
    Johanna Kliche, Dimitriya Hristoforova Garvanska, Leandro Simonetti, Dilip Badgujar, Doreen Dobritzsch, Jakob Nilsson, Norman E Davey, Ylva Ivarsson.
      Phosphorylation is a ubiquitous post-translation modification that regulates protein function by promoting, inhibiting or modulating protein-protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide-phage display library to screen for phosphosites that modulate short linear motif-based interactions. The peptidome covers ~13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild-type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif-mediated interactions. Affinity measurements confirmed the phospho-modulation of 14 out of 18 tested interactions. We performed a detailed follow-up on a phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP), demonstrating the essentiality of the phospho-dependency to the mitotic function of HURP. Structural characterisation of the clathrin-HURP complex elucidated the molecular basis for the phospho-dependency. Our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated interactions required for cellular function.
    Keywords:  clathrin; phage display; phosphomimetic mutation; phosphorylation; protein-protein interactions
    DOI:  https://doi.org/10.15252/msb.202211164
  6. Elife. 2023 May 25. pii: e85328. [Epub ahead of print]12
    The structural basis of the multi-step allosteric activation of Aurora B kinase.
    Dario Segura-Peña, Oda Hovet, Hemanga Gogoi, Jennine Dawicki-McKenna, Stine Malene Hansen Wøien, Manuel Carrer, Ben E Black, Michele Cascella, Nikolina Sekulic.
      Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.
    Keywords:  E. coli; biochemistry; chemical biology; molecular biophysics; structural biology; xenopus
    DOI:  https://doi.org/10.7554/eLife.85328
  7. Curr Protoc. 2023 May;3(5): e793
    Measurement of Microtubule Stability in Mammalian Cells.
    Carline Fermino do Rosário, Patricia Wadsworth.
      The microtubule cytoskeleton is essential for various biological processes such as the intracellular distribution of molecules and organelles, cell morphogenesis, chromosome segregation, and specification of the location of contractile ring formation. Distinct cell types contain microtubules with different extents of stability. For example, microtubules in neurons are highly stabilized to support organelle (or vesicular) transport over large distances, and microtubules in motile cells are more dynamic. In some cases, such as the mitotic spindle, both dynamic and stable microtubules coexist. Alteration of microtubule stability is connected to disease states, making understanding microtubule stability an important area of research. Methods to measure microtubule stability in mammalian cells are described here. Together, these approaches allow microtubule stability to be measured qualitatively or semiquantitatively following staining for post-translational modifications of tubulin or treating cells with microtubule destabilizing agents such as nocodazole. Microtubule stability can also be measured quantitatively by performing fluorescence recovery after photobleaching or fluorescence photoactivation of tubulin in live cells. These methods should be helpful for those seeking to understand microtubule dynamics and stabilization. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Fixing and staining cells for tubulin post-translational modifications Basic Protocol 2: Evaluating microtubule stability following treatment with nocodazole in live or fixed cells Basic Protocol 3: Measurement of microtubule dynamic turnover by quantification of fluorescence recovery after photobleaching Basic Protocol 4: Measurement of microtubule dynamic turnover by quantification of dissipation of fluorescence after photoactivation.
    Keywords:  FRAP; microtubule; nocodazole; photoactivation; post-translational modification
    DOI:  https://doi.org/10.1002/cpz1.793
  8. Eur J Cell Biol. 2023 May 19. pii: S0171-9335(23)00040-7. [Epub ahead of print]102(2): 151325
    Cockayne syndrome group A protein localizes at centrosomes during mitosis and regulates Cyclin B1 ubiquitination.
    Elena Paccosi, Giulia Artemi, Silvia Filippi, Alessio Balzerano, Federico Costanzo, Valentina Laghezza-Masci, Silvia Proietti, Luca Proietti-De-Santis.
      Mutations in CSA and CSB proteins cause Cockayne syndrome, a rare genetic neurodevelopment disorder. Alongside their demonstrated roles in DNA repair and transcription, these two proteins have recently been discovered to regulate cytokinesis, the final stage of the cell division. This last finding allowed, for the first time, to highlight an extranuclear localization of CS proteins, beyond the one already known at mitochondria. In this study, we demonstrated an additional role for CSA protein being recruited at centrosomes in a strictly determined step of mitosis, which ranges from pro-metaphase until metaphase exit. Centrosomal CSA exerts its function in specifically targeting the pool of centrosomal Cyclin B1 for ubiquitination and proteasomal degradation. Interestingly, a lack of CSA recruitment at centrosomes does not affect Cyclin B1 centrosomal localization but, instead, it causes its lasting centrosomal permanence, thus inducing Caspase 3 activation and apoptosis. The discovery of this unveiled before CSA recruitment at centrosomes opens a new and promising scenario for the understanding of some of the complex and different clinical aspects of Cockayne Syndrome.
    Keywords:  Apoptosis; Centrosome; Cockayne syndrome; Cyclin B1; Mitosis; Ubiquitination
    DOI:  https://doi.org/10.1016/j.ejcb.2023.151325
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