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1.
Mutat Res ; 854-855: 503197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660821

RESUMO

Telomeres, specialized structures at the ends of linear chromosomes, protect chromosome ends from degradation, recombination, and mis-repair. Critically short telomere length (TL) may result in chromosome instability (CIN), causing tumor promotion and, at higher levels, cell death and tumor suppression. Homocysteine (Hcy) is a sulfur-containing amino acid involved in one-carbon metabolism. Elevated plasma Hcy is a cancer risk factor. Human SH-SY5Y neuroblastoma cells were treated with pathophysiological concentrations of Hcy (15-120 µM) for 14 and 28 days. The cytokinesis-block micronucleus cytome assay was used to determine cytostasis (nuclear division index, NDI), cell death (apoptosis and necrosis), and CIN (micronuclei, nucleoplasmic bridges, and nuclear buds in binucleated cells). Quantitative PCR was used to measure TL and the expression of hTERT, the gene encoding the catalytic subunit of telomerase for TL elongation. The results showed that Hcy induced elongation of TL and fluctuating changes in expression of hTERT. TL elongation was associated with increased CIN. Hcy decreased the NDI and increased cell death. This study shows that there is cross-talk between Hcy and TL in tumor cells and supports the concept that high Hcy inhibits cell division and promotes the death of tumor cells by abnormal elongation of TL and elevation of CIN.


Assuntos
Instabilidade Cromossômica/genética , Homocisteína/genética , Neuroblastoma/genética , Telômero/genética , Apoptose/genética , Morte Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Citocinese/genética , Dano ao DNA/genética , Humanos , Micronúcleos com Defeito Cromossômico , Testes para Micronúcleos/métodos , Necrose/genética , Telomerase/genética
2.
Proc Natl Acad Sci U S A ; 117(31): 18511-18520, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690698

RESUMO

It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.


Assuntos
Actinas/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Divisão Celular , Chlamydomonas/química , Citocinese , Microtúbulos/metabolismo , Miosinas/química , Miosinas/metabolismo , Ligação Proteica
3.
PLoS Pathog ; 16(6): e1008587, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511279

RESUMO

New techniques for obtaining electron microscopy data through the cell volume are being increasingly utilized to answer cell biologic questions. Here, we present a three-dimensional atlas of Plasmodium falciparum ultrastructure throughout parasite cell division. Multiple wild type schizonts at different stages of segmentation, or budding, were imaged and rendered, and the 3D structure of their organelles and daughter cells are shown. Our high-resolution volume electron microscopy both confirms previously described features in 3D and adds new layers to our understanding of Plasmodium nuclear division. Interestingly, we demonstrate asynchrony of the final nuclear division, a process that had previously been reported as synchronous. Use of volume electron microscopy techniques for biological imaging is gaining prominence, and there is much we can learn from applying them to answer questions about Plasmodium cell biology. We provide this resource to encourage readers to consider adding these techniques to their cell biology toolbox.


Assuntos
Citocinese/fisiologia , Plasmodium falciparum , Humanos , Plasmodium falciparum/fisiologia , Plasmodium falciparum/ultraestrutura
4.
Nat Commun ; 11(1): 3231, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591511

RESUMO

Genomic instability (GI) predisposes cells to malignant transformation, however the molecular mechanisms that allow for the propagation of cells with a high degree of genomic instability remain unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells through the inhibition of RB1 and stimulator-of-interferon-genes (STING) to propagate cells with a high degree of GI. MiR-181a targeting of RB1 leads to profound nuclear defects and GI generating aberrant cytoplasmic DNA, however simultaneous miR-181a mediated inhibition of STING allows cells to bypass interferon mediated cell death. We also found that high miR-181a is associated with decreased IFNγ response and lymphocyte infiltration in patient tumors. DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation, thus, our findings are the first to identify a miRNA that can downregulate STING expression to suppress activation of intrinsic interferon signaling. This study introduces miR-181a as a putative biomarker and identifies the miR-181a-STING axis as a promising target for therapeutic exploitation.


Assuntos
Carcinogênese/genética , Transformação Celular Neoplásica/genética , Imunidade Inata/genética , MicroRNAs/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Núcleo Celular/patologia , Transformação Celular Neoplásica/patologia , Citocinese , Dano ao DNA , Células Epiteliais/patologia , Tubas Uterinas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Humanos , Interferons/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , MicroRNAs/genética , Mitose , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo
5.
PLoS Genet ; 16(5): e1008763, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32384077

RESUMO

The ACK family tyrosine kinase SID-3 is involved in the endocytic uptake of double-stranded RNA. Here we identified SID-3 as a previously unappreciated recycling regulator in the Caenorhabditis elegans intestine. The RAB-10 effector EHBP-1 is required for the endosomal localization of SID-3. Accordingly, animals with loss of SID-3 phenocopied the recycling defects observed in ehbp-1 and rab-10 single mutants. Moreover, we detected sequential protein interactions between EHBP-1, SID-3, NCK-1, and DYN-1. In the absence of SID-3, DYN-1 failed to localize at tubular recycling endosomes, and membrane tubules breaking away from endosomes were mostly absent, suggesting that SID-3 acts synergistically with the downstream DYN-1 to promote endosomal tubule fission. In agreement with these observations, overexpression of DYN-1 significantly increased recycling transport in SID-3-deficient cells. Finally, we noticed that loss of RAB-10 or EHBP-1 compromised feeding RNAi efficiency in multiple tissues, implicating basolateral recycling in the transport of RNA silencing signals. Taken together, our study demonstrated that in C. elegans intestinal epithelia, SID-3 acts downstream of EHBP-1 to direct fission of recycling endosomal tubules in concert with NCK-1 and DYN-1.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Citocinese/genética , Dinaminas/fisiologia , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Animais Geneticamente Modificados , Transporte Biológico/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Endocitose/genética , Estudo de Associação Genômica Ampla , Transdução de Sinais/genética , Proteínas rab de Ligação ao GTP/metabolismo
6.
Adv Exp Med Biol ; 1239: 233-244, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451862

RESUMO

Nearly five decades of research have established myosin as the main motor responsible for cytokinesis in organisms on the branch of the phylogenetic tree that includes amoebas, fungi and animals. This research has grown to be more mechanistic over the past decade, so we now have computer simulations of physically reasonable models that explain how myosins contribute to the assembly and constriction of contractile rings that pinch dividing cells into two daughter cells. Isoforms of myosin-II, from the same family as muscle myosins, are the main myosins for cytokinesis, but other myosins contribute to cytokinesis in fission yeast. Progress has been made on how animal cells use Rho-GTPases to control the accumulation and activity of myosin-II at the site of cleavage, but the regulatory mechanisms are less clear in other systems.


Assuntos
Citocinese , Miosinas , Animais , Filogenia , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(22): 12131-12142, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414923

RESUMO

Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.


Assuntos
Estruturas Cromossômicas/química , Cromossomos Humanos/química , DNA Topoisomerases Tipo II/metabolismo , Heterocromatina/química , Mitose , Segregação de Cromossomos , Estruturas Cromossômicas/genética , Cromossomos Humanos/genética , Citocinese , DNA Topoisomerases Tipo II/genética , Células HCT116 , Heterocromatina/genética , Humanos , Metáfase
8.
Mutat Res ; 850-851: 503143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247554

RESUMO

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.


Assuntos
Centrômero/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Testes de Mutagenicidade , Telômero/efeitos dos fármacos , Aneugênicos/farmacologia , Centrômero/genética , Citocinese/efeitos dos fármacos , Citocinese/genética , Dano ao DNA/genética , Humanos , Linfócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes para Micronúcleos , Mutagênicos/toxicidade , Medição de Risco , Telômero/genética
9.
Mutat Res ; 850-851: 503147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247562

RESUMO

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.


Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Fritillaria/química , Extratos Vegetais/farmacologia , Aurora Quinase B/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Instabilidade Cromossômica/genética , Colo/efeitos dos fármacos , Colo/patologia , Citocinese/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Extratos Vegetais/química , Raízes de Plantas/química , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a Y-Box/genética
10.
Nat Commun ; 11(1): 1746, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269212

RESUMO

In mammalian cell lines, the endosomal sorting complex required for transport (ESCRT)-III mediates abscission, the process that physically separates daughter cells and completes cell division. Cep55 protein is regarded as the master regulator of abscission, because it recruits ESCRT-III to the midbody (MB), the site of abscission. However, the importance of this mechanism in a mammalian organism has never been tested. Here we show that Cep55 is dispensable for mouse embryonic development and adult tissue homeostasis. Cep55-knockout offspring show microcephaly and primary neural progenitors require Cep55 and ESCRT for survival and abscission. However, Cep55 is dispensable for cell division in embryonic or adult tissues. In vitro, division of primary fibroblasts occurs without Cep55 and ESCRT-III at the midbody and is not affected by ESCRT depletion. Our work defines Cep55 as an abscission regulator only in specific tissue contexts and necessitates the re-evaluation of an alternative ESCRT-independent cell division mechanism.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Células Cultivadas , Córtex Cerebral/anormalidades , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Genótipo , Rim/anormalidades , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcefalia/patologia , Mitose
11.
Nat Commun ; 11(1): 1941, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321914

RESUMO

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Organelas/metabolismo , Sindecana-4/metabolismo , Sinteninas/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Membrana Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Organelas/genética , Ligação Proteica , Sindecana-4/genética , Sinteninas/genética
12.
Proc Natl Acad Sci U S A ; 117(17): 9584-9593, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32273386

RESUMO

Building a brain of the proper size and structure requires neural stem cells (NSCs) to divide with tight temporal and spatial control to produce different daughter cell types in proper numbers and sequence. Mammalian NSCs in the embryonic cortex must maintain their polarized epithelial structure as they undergo both early proliferative divisions and later neurogenic divisions. To do this, they undergo a polarized form of cytokinesis at the apical membrane that is not well understood. Here, we investigate whether polarized furrowing and abscission in mouse NSCs are regulated differently at earlier and later stages and in a cytokinesis mutant, Kif20b This mutant was previously shown to have microcephaly and elevated apoptosis of NSCs. We developed methods to live image furrow ingression and midbody abscission in NSCs within cortical explants. We find that polarized furrow ingression occurs at a steady rate and completes in ∼15 min at two different ages. However, ingression is slower in a subset of Kif20b mutant NSCs. Abscission is usually observed on both sides of the midbody and takes 65 to 75 min to complete. Surprisingly, abscission is accelerated in the Kif20b mutant NSCs. Postabscission midbody remnants are observed at the apical membranes of daughter cells and are much more abundant in early-stage cortices. After NSC divisions in vitro, midbody remnants are more often retained on the daughter cells of early proliferative divisions. Altogether, these results suggest that regulation of abscission timing and midbody remnants in embryonic NSCs may influence proper brain growth and structure.


Assuntos
Encéfalo/embriologia , Citocinese/fisiologia , Desenvolvimento Embrionário , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Animais , Anticorpos , Encéfalo/crescimento & desenvolvimento , Embrião de Mamíferos , Cinesina/genética , Cinesina/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação
13.
Nat Struct Mol Biol ; 27(4): 392-399, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251413

RESUMO

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.


Assuntos
Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Proteínas Oncogênicas/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Polimerização , Conformação Proteica
14.
PLoS Genet ; 16(4): e1008652, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32267837

RESUMO

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.


Assuntos
Divisão Celular/genética , Desenvolvimento Embrionário/genética , Herança Materna/genética , Mutação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Alelos , Animais , Blástula/citologia , Blástula/embriologia , Blástula/metabolismo , Padronização Corporal/genética , Núcleo Celular , Citocinese/genética , Feminino , Infertilidade Masculina/genética , Masculino , Mutagênese , Fenótipo , Proteínas de Peixe-Zebra/genética
15.
Yakugaku Zasshi ; 140(3): 391-393, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32115558

RESUMO

Recently, aging is becoming an important social problem in many developed countries including Japan. It is socially and universally important to unveil the impact of aging and extend healthy life expectancy. Here we show our recent finding that dedicator of cytokinesis 11 (DOCK11, also known as Zizimin2) may be involved in immunosenescence of B cells. DOCK11 was identified as a guanine nucleotide exchange factor for a small GTPase called cell division cycle 42. Expression of DOCK11 is restricted to lymphoid tissues, and becomes downregulated with age. Thus we examined the involvement of DOCK11 in immunosenescence of B-1a B cells as an example. B-1a cells are the main source of antibodies at steady state, and function as the first line of defense against infection. Although DOCK11 was expressed by B-1a cells, the expression levels declined with age. Furthermore, production of anti-pneumococcal immunoglobulin M antibodies was suppressed in aged mice, and was recovered by adoptive transfer with B-1a cells in a DOCK11-dependent manner. Thus DOCK11 may be involved in immunosenescence of B-1a cells.


Assuntos
Envelhecimento/imunologia , Imunossenescência , Animais , Linfócitos B/imunologia , Citocinese/imunologia , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imunoglobulina M , Camundongos , Estado Nutricional , Streptococcus pneumoniae/imunologia
16.
PLoS Genet ; 16(3): e1008685, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203516

RESUMO

Cell size is a complex trait, derived from both genetic and environmental factors. Environmental determinants of bacterial cell size identified to date primarily target assembly of cytosolic components of the cell division machinery. Whether certain environmental cues also impact cell size through changes in the assembly or activity of extracytoplasmic division proteins remains an open question. Here, we identify extracellular pH as a modulator of cell division and a significant determinant of cell size across evolutionarily distant bacterial species. In the Gram-negative model organism Escherichia coli, our data indicate environmental pH impacts the length at which cells divide by altering the ability of the terminal cell division protein FtsN to localize to the cytokinetic ring where it activates division. Acidic environments lead to enrichment of FtsN at the septum and activation of division at a reduced cell length. Alkaline pH inhibits FtsN localization and suppresses division activation. Altogether, our work reveals a previously unappreciated role for pH in bacterial cell size control.


Assuntos
Divisão Celular/fisiologia , Citocinese/fisiologia , Concentração de Íons de Hidrogênio , Proteínas de Bactérias/genética , Tamanho Celular , Parede Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Peptidoglicano/genética
17.
Proc Natl Acad Sci U S A ; 117(12): 6580-6589, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152126

RESUMO

Polarity decisions are central to many processes, including mitosis and chemotropism. In Saccharomyces cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converges on the small G protein Cdc42. However, pheromone-gradient sensing must override the Rsr1-dependent internal polarity cues used for budding. Using this model system, we asked what happens when intrinsic and extrinsic spatial cues are not aligned. Is there competition, or collaboration? By live-cell microscopy and microfluidics techniques, we uncovered three previously overlooked features of this signaling system. First, the cytokinesis-associated polarization patch serves as a polarity landmark independently of all known cues. Second, the Rax1-Rax2 complex functions as a pheromone-promoted polarity cue in the distal pole of the cells. Third, internal cues remain active during pheromone-gradient tracking and can interfere with this process, biasing the location of MPs. Yeast defective in internal-cue utilization align significantly better than wild type with artificially generated pheromone gradients.


Assuntos
Polaridade Celular , Quimiotaxia , Fator de Acasalamento/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Citocinese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
18.
Nat Commun ; 11(1): 612, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001712

RESUMO

The kinase Aurora B forms the chromosomal passenger complex (CPC) together with Borealin, INCENP, and Survivin to mediate chromosome condensation, the correction of erroneous spindle-kinetochore attachments, and cytokinesis. Phosphorylation of histone H3 Thr3 by Haspin kinase and of histone H2A Thr120 by Bub1 concentrates the CPC at the centromere. However, how the CPC is recruited to chromosome arms upon mitotic entry is unknown. Here, we show that asymmetric dimethylation at Arg2 on histone H3 (H3R2me2a) by protein arginine methyltransferase 6 (PRMT6) recruits the CPC to chromosome arms and facilitates histone H3S10 phosphorylation by Aurora B for chromosome condensation. Furthermore, in vitro assays show that Aurora B preferentially binds to the H3 peptide containing H3R2me2a and phosphorylates H3S10. Our findings indicate that the long-awaited key histone mark for CPC recruitment onto mitotic chromosomes is H3R2me2a, which is indispensable for maintaining appropriate CPC levels in dynamic translocation throughout mitosis.


Assuntos
Arginina/metabolismo , Aurora Quinase B/metabolismo , Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/patologia , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Citocinese , Desmetilação , Progressão da Doença , Feminino , Células HeLa , Histonas/química , Humanos , Células MCF-7 , Metilação , Mitose , Fosforilação , RNA Interferente Pequeno/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(8): 4169-4179, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32029597

RESUMO

Abscission is the terminal step of cytokinesis leading to the physical separation of the daughter cells. In response to the abnormal presence of lagging chromatin between dividing cells, an evolutionarily conserved abscission/NoCut checkpoint delays abscission and prevents formation of binucleated cells by stabilizing the cytokinetic intercellular bridge (ICB). How this bridge is stably maintained for hours while the checkpoint is activated is poorly understood and has been proposed to rely on F-actin in the bridge region. Here, we show that actin polymerization is indeed essential for stabilizing the ICB when lagging chromatin is present, but not in normal dividing cells. Mechanistically, we found that a cytosolic pool of human methionine sulfoxide reductase B2 (MsrB2) is strongly recruited at the midbody in response to the presence of lagging chromatin and functions within the ICB to promote actin polymerization there. Consistently, in MsrB2-depleted cells, F-actin levels are decreased in ICBs, and dividing cells with lagging chromatin become binucleated as a consequence of unstable bridges. We further demonstrate that MsrB2 selectively reduces oxidized actin monomers and thereby counteracts MICAL1, an enzyme known to depolymerize actin filaments by direct oxidation. Finally, MsrB2 colocalizes and genetically interacts with the checkpoint components Aurora B and ANCHR, and the abscission delay upon checkpoint activation by nuclear pore defects also depends on MsrB2. Altogether, this work reveals that actin reduction by MsrB2 is a key component of the abscission checkpoint that favors F-actin polymerization and limits tetraploidy, a starting point for tumorigenesis.


Assuntos
Actinas/metabolismo , Cromatina/metabolismo , Citocinese/fisiologia , Proteínas de Drosophila/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mitose/fisiologia , Animais , Linhagem Celular , Drosophila , Proteínas de Drosophila/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , Humanos , Metionina Sulfóxido Redutases/genética , Proteínas dos Microfilamentos/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredução
20.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118673, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32057919

RESUMO

Microtubules are polymers of α/ß-tubulin, with microtubule organization being regulated by microtubule-associated proteins (MAPs). Herein, we describe a novel role for the epithelial gene repressor, zinc finger E-box-binding homeobox 1 (ZEB1), that "switches" from a chromatin-associated protein during interphase, to a MAP that associates with α-, ß- and γ-tubulin during mitosis. Additionally, ZEB1 was also demonstrated to associate with γ-tubulin at the microtubule organizing center (MTOC). Using confocal microscopy, ZEB1 localization was predominantly nuclear during interphase, with α/ß-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during the stages of mitosis, ZEB1 co-localization with α-, ß-, and γ-tubulin was significantly increased, with the association commonly peaking during metaphase in multiple tumor cell-types. ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The increased interaction between ZEB1 and α-tubulin during mitosis was also confirmed using the proximity ligation assay. In contrast to ZEB1, its paralog ZEB2, was mainly perinuclear and cytoplasmic during interphase, showing some co-localization with α-tubulin during mitosis. Considering the association between ZEB1 with α/ß/γ-tubulin during mitosis, studies investigated ZEB1's role in the cell cycle. Silencing ZEB1 resulted in a G2-M arrest, which could be mediated by the up-regulation of p21Waf1/Cip1 and p27Kip1 that are known downstream targets repressed by ZEB1. However, it cannot be excluded the G2/M arrest observed after ZEB1 silencing is not due to its roles as a MAP. Collectively, ZEB1 plays a role as a MAP during mitosis and could be functionally involved in this process.


Assuntos
Cromatina/genética , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Pontos de Checagem do Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Citocinese/genética , Humanos , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica/genética , Fuso Acromático/genética , Tubulina (Proteína)/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/química
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