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BioRxiv : the Preprint Server For... Oct 2023During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each...
During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.
PubMed: 37905093
DOI: 10.1101/2023.06.29.547092 -
Nature Communications Sep 2023Accurate chromosome segregation in mitosis depends on multiprotein structures called kinetochores that are built on the centromeric region of sister chromatids and serve...
Accurate chromosome segregation in mitosis depends on multiprotein structures called kinetochores that are built on the centromeric region of sister chromatids and serve to capture mitotic spindle microtubules. In early mitosis, unattached kinetochores expand a crescent-shaped structure called fibrous corona whose function is to facilitate initial kinetochore-microtubule attachments and chromosome transport by microtubules. Subsequently, the fibrous corona must be timely disassembled to prevent segregation errors. Although recent studies provided new insights on the molecular content and mechanism of fibrous corona assembly, it remains unknown what triggers the disassembly of the outermost and dynamic layer of the kinetochore. Here, we show that Aurora A and B kinases phosphorylate CENP-E to release it from an autoinhibited state. At kinetochores, Aurora B phosphorylates CENP-E to prevent its premature removal together with other corona proteins by dynein. At the spindle poles, Aurora A phosphorylates CENP-E to promote chromosome congression and prevent accumulation of corona proteins at the centrosomes, allowing for their intracellular redistribution. Thus, we propose the Aurora A/B-CENP-E axis as a critical element of the long-sought-for mechanism of fibrous corona disassembly that is essential for accurate chromosome segregation.
Topics: Cell Nucleus Division; Centromere; Centrosome; Kinetochores; Spindle Apparatus; Humans
PubMed: 37658044
DOI: 10.1038/s41467-023-41091-2 -
Annual Review of Genomics and Human... Aug 2023In meiosis, homologous chromosome synapsis is mediated by a supramolecular protein structure, the synaptonemal complex (SC), that assembles between homologous chromosome... (Review)
Review
In meiosis, homologous chromosome synapsis is mediated by a supramolecular protein structure, the synaptonemal complex (SC), that assembles between homologous chromosome axes. The mammalian SC comprises at least eight largely coiled-coil proteins that interact and self-assemble to generate a long, zipper-like structure that holds homologous chromosomes in close proximity and promotes the formation of genetic crossovers and accurate meiotic chromosome segregation. In recent years, numerous mutations in human SC genes have been associated with different types of male and female infertility. Here, we integrate structural information on the human SC with mouse and human genetics to describe the molecular mechanisms by which SC mutations can result in human infertility. We outline certain themes in which different SC proteins are susceptible to different types of disease mutation and how genetic variants with seemingly minor effects on SC proteins may act as dominant-negative mutations in which the heterozygous state is pathogenic.
Topics: Male; Female; Humans; Mice; Animals; Synaptonemal Complex; Chromosome Pairing; Meiosis; Infertility; Mutation; Mammals
PubMed: 37159901
DOI: 10.1146/annurev-genom-110122-090239 -
Nature Genetics Sep 2023Modified parental histones are segregated symmetrically to daughter DNA strands during replication and can be inherited through mitosis. How this may sustain the...
Modified parental histones are segregated symmetrically to daughter DNA strands during replication and can be inherited through mitosis. How this may sustain the epigenome and cell identity remains unknown. Here we show that transmission of histone-based information during DNA replication maintains epigenome fidelity and embryonic stem cell plasticity. Asymmetric segregation of parental histones H3-H4 in MCM2-2A mutants compromised mitotic inheritance of histone modifications and globally altered the epigenome. This included widespread spurious deposition of repressive modifications, suggesting elevated epigenetic noise. Moreover, H3K9me3 loss at repeats caused derepression and H3K27me3 redistribution across bivalent promoters correlated with misexpression of developmental genes. MCM2-2A mutation challenged dynamic transitions in cellular states across the cell cycle, enhancing naïve pluripotency and reducing lineage priming in G1. Furthermore, developmental competence was diminished, correlating with impaired exit from pluripotency. Collectively, this argues that epigenetic inheritance of histone modifications maintains a correctly balanced and dynamic chromatin landscape able to support mammalian cell differentiation.
Topics: Animals; Histones; Epigenome; Chromatin; Embryonic Stem Cells; Mitosis; Mammals
PubMed: 37666988
DOI: 10.1038/s41588-023-01476-x -
Science Advances Aug 2023Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of...
Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.
Topics: Rad51 Recombinase; DNA Repair; Proteins; DNA; Mitosis
PubMed: 37556550
DOI: 10.1126/sciadv.adf4082 -
Cell Death & Disease Jul 2023Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein...
Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein MAD2, leading to mitotic arrest and cell death. However, some tumor cells are capable of exiting mitosis and consequently increasing CIN. How cells escape the mitotic arrest induced by MAD2 and proliferate with CIN is not well understood. Here, we explored loss-of-function screens and drug sensitivity tests associated with MAD2 levels in aneuploid cells and identified that aneuploid cells with high MAD2 levels are more sensitive to FOXM1 depletion. Inhibition of FOXM1 promotes MAD2-mediated mitotic arrest and exacerbates CIN. Conversely, elevating FOXM1 expression in MAD2-overexpressing human cell lines reverts prolonged mitosis and rescues mitotic errors, cell death and proliferative disadvantages. Mechanistically, we found that FOXM1 facilitates mitotic exit by inhibiting the spindle assembly checkpoint (SAC) and the expression of Cyclin B. Notably, we observed that FOXM1 is upregulated upon aneuploid induction in cells with dysfunctional SAC and error-prone mitosis, and these cells are sensitive to FOXM1 knockdown, indicating a novel vulnerability of aneuploid cells.
Topics: Humans; Cell Cycle Proteins; Mad2 Proteins; Mitosis; Cell Line, Tumor; Spindle Apparatus; Aneuploidy; Forkhead Box Protein M1
PubMed: 37452072
DOI: 10.1038/s41419-023-05946-2 -
The EMBO Journal Jul 2023During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of...
During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organization of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.
Topics: Chromosome Segregation; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Protein Binding; Animals
PubMed: 37203876
DOI: 10.15252/embj.2022112504 -
Cell Reports Dec 2023Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence...
Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles and then enter a CDK2-low quiescence, whereas lysine withdrawal causes immediate cell-cycle stalling. Methionine withdrawal triggers a restriction point phenotype similar to serum starvation or Mek inhibition: upon methionine withdrawal, cells complete their current cell cycle and enter a CDK2-low quiescence after mitosis. Modulation of restriction point regulators p21/p27 or cyclin D1 enables short-term rescue of proliferation under methionine and leucine withdrawal, and to a lesser extent lysine withdrawal, revealing a checkpoint connecting nutrient signaling to cell-cycle entry.
Topics: Cell Cycle Proteins; Cyclin-Dependent Kinases; Cyclin-Dependent Kinase Inhibitor p21; Amino Acids; Leucine; Lysine; Cell Cycle; Cyclin-Dependent Kinase 2; Cell Cycle Checkpoints; Mitosis; Methionine; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase Inhibitor p27
PubMed: 38070134
DOI: 10.1016/j.celrep.2023.113539 -
BioRxiv : the Preprint Server For... Aug 2023Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. Mitotic entry is initiated by increased phosphorylation of mitotic...
Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases, while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55. While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited. For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent) and FAM122A (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.
PubMed: 37693408
DOI: 10.1101/2023.08.31.555365 -
ELife Dec 2023During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each...
During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.
Topics: Spindle Apparatus; Kinetochores; Microtubules; Mitosis; Chromosome Segregation
PubMed: 38150374
DOI: 10.7554/eLife.89467