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Current Biology : CB Jan 2021Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact...
Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination, and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganization of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that bind to centromeres in the absence of the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that the Ctf19c inner kinetochore complex is essential for kinetochore organization in meiosis. Our functional analyses identify a Ctf19c-dependent kinetochore assembly pathway that is dispensable for mitotic growth but becomes critical upon meiotic entry. Therefore, changes in kinetochore composition and a distinct assembly pathway specialize meiotic kinetochores for successful gametogenesis.
Topics: Centromere; Chromatin; Chromosome Segregation; Cytoskeletal Proteins; Kinetochores; Meiosis; Mitosis; Proteomics; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33157029
DOI: 10.1016/j.cub.2020.10.025 -
The Journal of Cell Biology Nov 2020During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome...
During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid-binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A-RNA complexes on mitotic chromosomes, defects in metaphase chromosome alignment, and elevated rates of chromosome missegregation in anaphase. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase and demonstrates that Aurora-B-dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.
Topics: Aurora Kinase B; Cell Nucleus; Chromatin; Chromosomes, Human; HEK293 Cells; Heterogeneous-Nuclear Ribonucleoprotein U; Humans; Mitosis; Phosphorylation; RNA
PubMed: 33053167
DOI: 10.1083/jcb.201910148 -
Biochemistry. Biokhimiia Sep 2020We investigated distribution and functions of beta- and gamma-cytoplasmic actins (CYAs) at different stages of non-neoplastic epithelial cell division using laser...
We investigated distribution and functions of beta- and gamma-cytoplasmic actins (CYAs) at different stages of non-neoplastic epithelial cell division using laser scanning microscopy (LSM). Here, we demonstrated that beta- and gamma-CYAs are spatially segregated in the early prophase, anaphase, telophase, and cytokinesis. Small interfering RNA (siRNA) experiments revealed that in both beta-CYA- and gamma-CYA-depleted cells, the number of cells was significantly reduced compared with the siRNA controls. Beta-CYA depletion resulted in an enlargement of the cell area in metaphase and high percentage of polynuclear cells compared with the siRNA control, indicating a potential failure of cytokinesis. Gamma-CYA depletion resulted in a reduced percentage of mitotic cells. We also observed the interdependence between the actin isoforms and the microtubule system in mitosis: (i) a decrease in the gamma-CYA led to impaired mitotic spindle organization; (ii) suppression of tubulin polymerization caused impaired beta-CYA reorganization, as incubation with colcemid blocked the transfer of short beta-actin polymers from the basal to the cortical compartment. We conclude that both actin isoforms are essential for proper cell division, but each isoform has its own specific functional role in this process.
Topics: Actins; Cells, Cultured; Cytokinesis; Cytoplasm; Humans; Keratinocytes; Microtubules; Mitosis; Protein Isoforms
PubMed: 33050852
DOI: 10.1134/S0006297920090072 -
Communications Biology Sep 2020Meiosis produces four haploid cells after two successive divisions in sexually reproducing organisms. A critical event during meiosis is construction of the synaptonemal...
Meiosis produces four haploid cells after two successive divisions in sexually reproducing organisms. A critical event during meiosis is construction of the synaptonemal complex (SC), a large, protein-based bridge that physically links homologous chromosomes. The SC facilitates meiotic recombination, chromosome compaction, and the eventual separation of homologous chromosomes at metaphase I. We present experiments directly measuring physical properties of captured mammalian meiotic prophase I chromosomes. Mouse meiotic chromosomes are about ten-fold stiffer than somatic mitotic chromosomes, even for genetic mutants lacking SYCP1, the central element of the SC. Meiotic chromosomes dissolve when treated with nucleases, but only weaken when treated with proteases, suggesting that the SC is not rigidly connected, and that meiotic prophase I chromosomes are a gel meshwork of chromatin, similar to mitotic chromosomes. These results are consistent with a liquid- or liquid-crystal SC, but with SC-chromatin stiff enough to mechanically drive crossover interference.
Topics: Animals; Chromatin; Chromosomes, Mammalian; Fluorescent Antibody Technique; Gels; Male; Meiotic Prophase I; Mice; Mice, Inbred C57BL; Spermatocytes
PubMed: 32999386
DOI: 10.1038/s42003-020-01265-w -
Frontiers in Oncology 2020Epidermal growth factor receptor major driver mutations may affect downstream molecular networks and pathways, which would influence treatment outcomes of non-small...
Epidermal growth factor receptor major driver mutations may affect downstream molecular networks and pathways, which would influence treatment outcomes of non-small cell lung cancer (NSCLC). This study aimed to unveil profiles of mutant proteins expressed in lung adenocarcinomas of 36 patients harboring representative driver mutations (Ex19del, nine; L858R, nine; no Ex19del/L858R, 18). Surprisingly, the orthogonal partial least squares discriminant analysis performed for identified mutant proteins demonstrated the profound differences in distance among the different mutation groups, suggesting that cancer cells harboring L858R or Ex19del emerge from cellular origins different from L858R/Ex19del-negative cells. Weighted gene coexpression network analysis, together with over-representative analysis, identified 18 coexpressed modules and their eigen proteins. Pathways enriched differentially for both the L858R and Ex19del mutations included carboxylic acid metabolic process, cell cycle, developmental biology, cellular responses to stress, mitotic prophase, cell proliferation, growth, epithelial to mesenchymal transition (EMT), and immune system. The IPA causal network analysis identified the highly activated networks of , and under the L858R mutation, whereas those of , and were highly activated under the Ex19del mutation. Interestingly, the downregulated causal network of osimertinib intervention showed the highest significance in overlap -value among most causal networks predicted under the L858R mutation. We also identified the causal network of MAPK interacting serine/threonine kinase 1/2 () highly activated differentially under the L858R mutation. Tumor-suppressor , a component of the Hippo pathways, was highly inhibited commonly under both L858R and Ex19del mutations. Our results could identify disease-related protein molecular networks from the landscape of single amino acid variants. Our findings may help identify potential therapeutic targets and develop therapeutic strategies to improve patient outcomes.
PubMed: 32983988
DOI: 10.3389/fonc.2020.01494 -
Molecular Biology of the Cell Oct 2020NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and...
NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding-deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindle-independent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA.
Topics: Antigens, Nuclear; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Proteins; Cell Nucleus; Cell Nucleus Division; Chromatin; Chromosomal Proteins, Non-Histone; Chromosomes; DNA; HeLa Cells; Humans; Mitosis; Nuclear Matrix-Associated Proteins; Nuclear Proteins; Spindle Apparatus
PubMed: 32845810
DOI: 10.1091/mbc.E20-06-0415 -
Journal of Toxicology 2020Appropriate effluent treatment processes are expected to significantly reduce the toxicity of effluents before they are released to the natural environment. The present...
Appropriate effluent treatment processes are expected to significantly reduce the toxicity of effluents before they are released to the natural environment. The present study was aimed to assess the spatial and temporal variations of the physical and chemical water quality parameters of a natural water body receiving treated textile effluents and to assess the chromosomal abnormalities induced by the treated textile effluents. Four sampling sites (A: effluent discharge point; B: 100 m downstream from site A along the tributary; C: 200 m downstream from site A along the tributary; D: 100 m upstream from site A along the tributary) were selected associated to a tributary that received treated textile effluent. The physical and chemical water quality parameters were measured in the composite water samples collected from the study sites, and bioassay was conducted using aged tap water as the control. Sampling was conducted in both rainy and dry seasons. The conductivity, TDS, COD, and colour intensity of the water samples collected from the study sites were significantly higher during the dry season compared to those in the rainy season. root meristematic cells exposed to water samples from sites A, B, and C showed a significantly high interphase and prophase indices compared to those exposed to aged tap water and upstream site during both rainy and dry seasons. The mitotic index of the root tip cells of bulbs exposed to the water samples collected from the effluent discharge point (site A) and from the 100 m downstream site from site A (site B) was significantly lower than that of the other sites in both rainy and dry seasons. However, the mitotic index of the root tip cells of bulbs exposed to the water samples from the upstream site was not significantly different from that of the control treatment during both sampling seasons. The bioassay indicated that the mitotic index and phase index of the root meristematic cells of can be affected by the treated textile effluents released to the water body and the occurrence of C metaphase, chromosomal adherence, bridges, disturbed anaphase, vagrant chromosomes, and chromosomal breaks indicated that the treated textile effluent receiving tributary can possibly contain genotoxic and mutagenic compounds which can induce chromosomal abnormalities.
PubMed: 32831832
DOI: 10.1155/2020/8814196 -
Essays in Biochemistry Sep 2020While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of... (Review)
Review
While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.
Topics: Animals; Centromere; Chromosomal Proteins, Non-Histone; Drosophila; HeLa Cells; Humans; Kinetochores; Meiosis; Mice
PubMed: 32794572
DOI: 10.1042/EBC20190080 -
The EMBO Journal Oct 2020Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with...
Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.
Topics: Binding Sites; Catalytic Domain; Cell Cycle Proteins; Chromosome Pairing; DNA Helicases; DNA Repair; DNA Repair Enzymes; DNA Topoisomerases; DNA, Single-Stranded; DNA-Binding Proteins; Mutation; Protein Binding; Protein Domains; Rad51 Recombinase; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32790929
DOI: 10.15252/embj.2020105705 -
Molecular Cell Sep 2020A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of...
A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.
Topics: Adenosine Triphosphatases; Anaphase; Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosomes; DNA Topoisomerases, Type II; DNA-Binding Proteins; Imaging, Three-Dimensional; Mammals; Metaphase; Mitosis; Prophase
PubMed: 32768407
DOI: 10.1016/j.molcel.2020.07.002