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PLoS Genetics Jan 2023The Arp2/3 complex is an actin nucleator with well-characterized activities in cell morphogenesis and movement, but its roles in nuclear processes are relatively...
The Arp2/3 complex is an actin nucleator with well-characterized activities in cell morphogenesis and movement, but its roles in nuclear processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity and cell cycle progression using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 complex ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Micronuclei arise in ArpC2 iKO cells due to chromatin segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged DNA fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. In the nuclei of Arp2/3-depleted cells, the tumor suppressor p53 is activated and the cell cycle inhibitor Cdkn1a/p21 mediates a G1 arrest. In the cytosol, micronuclei are recognized by the DNA sensor cGAS, which is important for stimulating a STING- and IRF3-associated interferon response. These studies establish functional requirements for the mammalian Arp2/3 complex in mitotic spindle organization and genome stability. They also expand our understanding of the mechanisms leading to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.
Topics: Animals; Mice; Actin-Related Protein 2-3 Complex; Actins; Cellular Senescence; DNA; Genomic Instability; Mitosis
PubMed: 36706133
DOI: 10.1371/journal.pgen.1010045 -
Science Advances Jan 2023Although mitotic chromosomes are highly compacted and transcriptionally inert, some active chromatin features are retained during mitosis to ensure the proper...
Although mitotic chromosomes are highly compacted and transcriptionally inert, some active chromatin features are retained during mitosis to ensure the proper postmitotic reestablishment of maternal transcriptional programs, a phenomenon termed "mitotic bookmarking." However, the dynamics and regulation of mitotic bookmarking have not been systemically surveyed. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we examined 6538 mitotic L02 human liver cells of variable stages and found that chromatin accessibility remained changing throughout cell division, with a constant decrease until metaphase and a gradual increase as chromosomes segregated. In particular, a subset of chromatin regions were identified to remain open throughout mitosis, and genes associated with these bookmarked regions are primarily linked to rapid reactivation upon mitotic exit. We also demonstrated that nuclear transcription factor Y subunit α (NF-YA) preferentially occupied bookmarked regions and contributed to transcriptional reactivation after mitosis. Our study uncovers the dynamic and regulatory blueprint of mitotic bookmarking.
Topics: Humans; Chromatin; Chromosomes; Transcription Factors; Mitosis
PubMed: 36696508
DOI: 10.1126/sciadv.add2175 -
Cytogenetic and Genome Research 2022Mitotic chromosomes of butterflies, which look like dots or short filaments in most published data, are generally considered to lack localised centromeres and thus to be...
Mitotic chromosomes of butterflies, which look like dots or short filaments in most published data, are generally considered to lack localised centromeres and thus to be holokinetic. This particularity, observed in a number of other invertebrates, is associated with meiotic particularities known as "inverted meiosis," in which the first division is equational, i.e., centromere splitting-up and segregation of sister chromatids instead of homologous chromosomes. However, the accurate analysis of butterfly chromosomes is difficult because (1) their size is very small, equivalent to 2 bands of a mammalian metaphase chromosome, and (2) they lack satellite DNA/heterochromatin in putative centromere regions and therefore marked primary constrictions. Our improved conditions for basic chromosome preparations, here applied to 6 butterfly species belonging to families Nymphalidae and Pieridae challenges the holocentricity of their chromosomes: in spite of the absence of primary constrictions, sister chromatids are recurrently held together at definite positions during mitotic metaphase, which makes possible to establish karyotypes composed of acrocentric and submetacentric chromosomes. The total number of chromosomes per karyotype is roughly inversely proportional to that of non-acrocentric chromosomes, which suggests the occurrence of frequent robertsonian-like fusions or fissions during evolution. Furthermore, the behaviour and morphological changes of chromosomes along the various phases of meiosis do not seem to differ much from those of canonical meiosis. In particular, at metaphase II chromosomes clearly have 2 sister chromatids, which refutes that anaphase I was equational. Thus, we propose an alternative mechanism to holocentricity for explaining the large variations in chromosome numbers in butterflies: (1) in the ancestral karyotype, composed of about 62 mostly acrocentric chromosomes, the centromeres, devoid of centromeric heterochromatin/satellite DNA, were located at contact with telomeric heterochromatin; (2) the instability of telomeric heterochromatin largely contributed to drive the multiple rearrangements, principally chromosome fusions, which occurred during butterfly evolution.
Topics: Humans; Animals; Butterflies; Heterochromatin; DNA, Satellite; Chromosomes; Centromere; Meiosis; Chromatids; Karyotyping; Mammals
PubMed: 36689925
DOI: 10.1159/000526034 -
Genes Jan 2023Amongst the 460 karyotypes of Polyphagan Coleoptera that we studied, 50 (10.8%) were carriers of an X autosome rearrangement. In addition to mitotic metaphase analysis,...
Amongst the 460 karyotypes of Polyphagan Coleoptera that we studied, 50 (10.8%) were carriers of an X autosome rearrangement. In addition to mitotic metaphase analysis, the correct diagnosis was performed on meiotic cells, principally at the pachytene stage. The percentages of these inter-chromosomal rearrangements, principally fusions, varied in relation to the total diploid number of chromosomes: high (51%) below 19, null at 19, low (2.7%) at 20 (the ancestral and modal number), and slightly increasing from 7.1% to 16.7% from 22 to above 30. The involvement of the X in chromosome fusions appears to be more than seven-fold higher than expected for the average of the autosomes. Examples of karyotypes with X autosome rearrangements are shown, including insertion of the whole X in the autosome (ins(A;X)), which has never been reported before in animals. End-to-end fusions (Robertsonian translocations, terminal rearrangements, and pseudo-dicentrics) are the most frequent types of X autosome rearrangements. As in the 34 species with a 19,X formula, there was no trace of the Y chromosome in the 50 karyotypes with an X autosome rearrangement, which demonstrates the dispensability of this chromosome. In most instances, C-banded heterochromatin was present at the X autosome junction, which suggests that it insulates the gonosome from the autosome portions, whose genes are subjected to different levels of expression. Finally, it is proposed that the very preferential involvement of the X in inter-chromosome rearrangements is explained by: (1) the frequent acrocentric morphology of the X, thus the terminal position of constitutive heterochromatin, which can insulate the attached gonosomal and autosomal components; (2) the dispensability of the Y chromosome, which considerably minimizes the deleterious consequences of the heterozygous status in male meiosis, (3) following the rapid loss of the useless Y chromosome, the correct segregation of the X autosome-autosome trivalent, which ipso facto is ensured by a chiasma in its autosomal portion.
Topics: Animals; Male; X Chromosome; Heterochromatin; Coleoptera; Y Chromosome; Sex Chromosomes
PubMed: 36672891
DOI: 10.3390/genes14010150 -
EMBO Reports Mar 2023The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily...
The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Pins, Gαi, Mud, and Dynein in flies - that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In this study we revisit the canonical model and find that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over-ridden by interaction with another Pins interacting protein, Inscuteable.
Topics: Animals; Drosophila; Drosophila Proteins; Cell Cycle Proteins; Cell Division; Spindle Apparatus; Microtubules
PubMed: 36629398
DOI: 10.15252/embr.202256074 -
Current Biology : CB Feb 2023Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements. To prevent micronuclei formation in mitosis, the dynamic plus ends of...
Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements. To prevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes, whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes. The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC). However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments, detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.
Topics: Humans; Centrosome; Chromosome Segregation; Kinetochores; Microtubules; Mitosis; Spindle Apparatus; Spindle Poles
PubMed: 36626904
DOI: 10.1016/j.cub.2022.12.017 -
Plants (Basel, Switzerland) Dec 2022Green synthesis of nanoparticles is receiving more attention these days since it is simple to use and prepare, uses fewer harsh chemicals and chemical reactions, and is...
Green synthesis of nanoparticles is receiving more attention these days since it is simple to use and prepare, uses fewer harsh chemicals and chemical reactions, and is environmentally benign. A novel strategy aims to recycle poisonous plant chemicals and use them as natural stabilizing capping agents for nanoparticles. In this investigation, silver nanoparticles loaded with latex from L. (Cy-AgNPs) were examined using a transmission electron microscope, FT-IR spectroscopy, and UV-visible spectroscopy. Additionally, using as a model test plant, the genotoxicity and cytotoxicity effects of crude latex and various concentrations of Cy-AgNPs were studied. The majority of the particles were spherical in shape. The highest antioxidant activity using DPPH was illustrated for CAgNPs (25 mg/L) (70.26 ± 1.32%) and decreased with increased concentrations of Cy-AGNPs. Antibacterial activity for all treatments was determined showing that the highest antibacterial activity was for Cy-AgNPs (50 mg/L) with inhibition zone 24 ± 0.014 mm against , 19 ± 0.12 mm against , and 23 ± 0.015 against . For phytochemical analysis, the highest levels of secondary metabolites from phenolic content, flavonoids, tannins, and alkaloids, were found in Cy-AgNPs (25 mg/L). treated with Cy-AgNPs- (25 mg/L) displayed the highest mitotic index (MI%) value of 9.08% compared to other Cy-AgNP concentrations (50-100 mg/L) and crude latex concentrations (3%). To detect cytotoxicity, a variety of chromosomal abnormalities were used, including micronuclei at interphase, disturbed at metaphase and anaphase, chromosomal stickiness, bridges, and laggards. The concentration of Cy-AgNPs (25 mg/L) had the lowest level of chromosomal aberrations, with a value of 23.41% versus 20.81% for the control. Proteins from seeds treated with produced sixteen bands on SDS-PAGE, comprising ten monomorphic bands and six polymorphic bands, for a total percentage of polymorphism of 37.5%. Eight ISSR primers were employed to generate a total of 79 bands, 56 of which were polymorphic and 23 of which were common. Primer ISSR 14 has the highest level of polymorphism (92.86%), according to the data. Using biochemical SDS-PAGE and ISSR molecular markers, Cy-AgNPs (25 mg/L) showed the highest percentage of genomic template stability (GTS%), with values of 80% and 51.28%, respectively. The findings of this work suggest employing CyAgNPs (25 mg/L) in pharmaceutical purposes due to its highest content of bioactive compounds and lowest concentration of chromosomal abnormalities.
PubMed: 36616301
DOI: 10.3390/plants12010172 -
Animals : An Open Access Journal From... Dec 2022This brief review is focused on the viviparous lizard (Lichtenstein, 1823), of the family Lacertidae, which possesses female heterogamety and multiple sex chromosomes... (Review)
Review
A Brief Review of Meiotic Chromosomes in Early Spermatogenesis and Oogenesis and Mitotic Chromosomes in the Viviparous Lizard (Squamata: Lacertidae) with Multiple Sex Chromosomes.
This brief review is focused on the viviparous lizard (Lichtenstein, 1823), of the family Lacertidae, which possesses female heterogamety and multiple sex chromosomes (male 2 = 36, ZZZZ/ZZW, female 2 = 35, with variable W sex chromosome). Multiple sex chromosomes and their changes may influence meiosis and the female meiotic drive, and they may play a role in reproductive isolation. In two cryptic taxa of with different W sex chromosomes, meiosis during early spermatogenesis and oogenesis proceeds normally, without any disturbances, with the formation of haploid spermatocytes, and in female meiosis with the formation of synaptonemal complexes (SCs) and the lampbrush chromosomes. In females, the SC number was constantly equal to 19 (according to the SC length, 16 SC autosomal bivalents plus three presumed SC sex chromosome elements). No variability in the chromosomes at the early stages of meiotic prophase I, and no significant disturbances in the chromosome segregation at the anaphase-telophase I stage, have been discovered, and haploid oocytes ( = 17) at the metaphase II stage have been revealed. There should be a factor/factors that maintain the multiple sex chromosomes, their equal transmission, and the course of meiosis in these cryptic forms of
PubMed: 36611629
DOI: 10.3390/ani13010019 -
Cell & Bioscience Dec 2022ADD1 (adducin-1) and TPX2 (targeting protein for Xklp2) are centrosomal proteins and regulate mitotic spindle assembly. Mammalian oocytes that segregate homologous...
BACKGROUND
ADD1 (adducin-1) and TPX2 (targeting protein for Xklp2) are centrosomal proteins and regulate mitotic spindle assembly. Mammalian oocytes that segregate homologous chromosomes in Meiosis I and sister chromatids in Meiosis II with a spindle lacking centrosomes are more prone to chromosome segregation errors than in mitosis. However, the regulatory mechanisms of oocyte spindle assembly and the functions of ADD1 and TPX2 in this process remain elusive.
RESULT
We found that the expression levels and localization of ADD1, S726 phosphorylated ADD1 (p-ADD1), and TPX2 proteins exhibited spindle assembly-dependent dynamic changes during mouse oocyte meiosis. Taxol treatment, which stabilizes the microtubule polymer and protects it from disassembly, made the signals of ADD1, p-ADD1, and TPX2 present in the microtubule organizing centers of small asters and spindles. Knockdown of approximately 60% of ADD1 protein levels destabilized interpolar microtubules in the meiotic spindle, resulting in aberrant chromosome alignment, reduced first polar body extrusion, and increased aneuploidy in metaphase II oocytes, but did not affect K-fiber homeostasis and the expression and localization of TPX2. Strikingly, TPX2 deficiency caused increased protein content of ADD1, but decreased expression and detachment of p-ADD1 from the spindle, thereby arresting mouse oocytes at the metaphase I stage with collapsed spindles.
CONCLUSION
Phosphorylation of ADD1 at S726 by TPX2 mediates acentriolar spindle assembly and precise chromosome segregation in mouse oocytes.
PubMed: 36539904
DOI: 10.1186/s13578-022-00943-y -
Communications Biology Dec 2022Methodologies for direct intracellular imaging of RNA and DNA are necessary for the advancement of bioimaging. Here we show direct label-free imaging of RNA and DNA in...
Methodologies for direct intracellular imaging of RNA and DNA are necessary for the advancement of bioimaging. Here we show direct label-free imaging of RNA and DNA in single cells by isolating their accurate Raman spectra. Raman images of DNA from interphase cells show intact nucleus, while those from mitotic cells reveal condensed chromosome. The condensed chromosome images are accurate enough to assign the stage of mitotic cell division (e.g., metaphase). Raman spectral features indicate B-DNA double helical conformational form in all the cell lines investigated here. The Raman images of RNAs, on the other hand, reveal liquid-liquid phase separated (LLPS) membraneless organelles in interphase cells, which disappears during mitosis. Further, the Raman spectrum of proteins from the intracellular LLPS organelles indicates slight enrichment of amyloid-like secondary structural features. Vibrational imaging of intracellular DNA and RNA simultaneously would open myriad of opportunities for examining functional biochemical aspects of cells and organelles.
Topics: RNA; Biomolecular Condensates; Cell Nucleus; DNA; Mitosis
PubMed: 36528668
DOI: 10.1038/s42003-022-04342-4