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/genéticaRESUMO
Meiotic cytokinesis influences the fertility and ploidy of gametes. However, limited information is available on the genetic control of meiotic cytokinesis in plants. Here, we identified a rice mutant with low male fertility, defective callose in meiosis 1 (dcm1). The pollen grains of dcm1 are proved to be defective in exine formation. Meiotic cytokinesis is disrupted in dcm1, resulting in disordered spindle orientation during meiosis II and formation of pollen grains with varied size and DNA content. We demonstrated that meiotic cytokinesis defect in dcm1 is caused by prematurely dissolution of callosic plates. Furthermore, peripheral callose surrounding the dcm1 pollen mother cells (PMCs) also disappeared untimely around pachytene. The DCM1 protein contains five tandem CCCH motifs and interacts with nuclear poly (A) binding proteins (PABNs) in nuclear speckles. The expression profiles of genes related to callose synthesis and degradation are significantly modified in dcm1. Together, we propose that DCM1 plays an essential role in male meiotic cytokinesis by preserving callose from prematurely dissolution in rice.
Assuntos
Glucanos/metabolismo , Oryza/citologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Citocinese/genética , Citocinese/fisiologia , Técnicas de Inativação de Genes , Genes de Plantas , Meiose/genética , Meiose/fisiologia , Oryza/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/citologia , Pólen/genética , Pólen/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Homologia de Sequência de Aminoácidos , Sequências de Repetição em Tandem , Dedos de Zinco/genéticaRESUMO
Previously we have shown that low temperature stress in Arabidopsis causes defects in microtubule organization and cytokinesis in male meiocytes, which leads to the formation of diploid pollen. Because cytokinin (CK) mediates multiple physiological responses to cold stress, we investigated whether CK signaling is involved in cold-induced diploid pollen formation. To this end, we monitored male sporogenesis in a series of mutants defective in CK metabolism and signalling. Arabidopsis plants with altered CK homeostasis, that is, the ahk2-2 ahk3-3 double and the ahp2-1 ahp3 ahp5-2 triple mutant, were cold sensitive and displayed similar defective male meiotic cytokinesis as wild type plants upon cold stress. These findings demonstrate that the AHK2/3-AHP2/3/5 CK-signaling module is not required for cold-induced ploidy stability of male gamete in Arabidopsis. Cytological analysis further revealed that the cold-induced cytokinesis defects in the ahk2-2 ahk3-3 mutant correlated with irregular organization of the radial microtubule array (RMA) in tetrad microspores at the end of male meiosis. Contrary to the ahk and ahp mutants, Arabidopsis plants defective for ARR1, a downstream target of ahk and ahp mediated CK signalling, displayed higher cold-tolerance of male meiotic cytokinesis program. We here suggest that the transcription regulator ARR1 may act independently from the CK AHK2/3-AHP2/3/5 signaling module in conveying the cold response to male meiocytes.
Assuntos
Citocinese/genética , Citocinese/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Temperatura Baixa , Citocininas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Histidina Quinase/genética , Histidina Quinase/metabolismo , Meiose/fisiologia , Mutação , Fosfotransferases/metabolismo , Pólen/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Esporos Fúngicos/metabolismo , Estresse FisiológicoRESUMO
Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/metabolismo , Exocitose , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Antioxidantes/farmacologia , Hidrolases de Éster Carboxílico/genética , Catequina/análogos & derivados , Catequina/farmacologia , Divisão Celular/genética , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/genética , Citocinese/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Microscopia Imunoeletrônica , Proteínas de Plantas/genética , Pólen/citologia , Pólen/metabolismo , Transporte Proteico/efeitos dos fármacos , Via Secretória , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Nicotiana/citologia , Nicotiana/genética , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures-openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores-the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ploidias , Pólen/genética , Arabidopsis/crescimento & desenvolvimento , Parede Celular/genética , Citocinese/genética , Meiose/genética , Proteínas Mutantes/genética , Pólen/crescimento & desenvolvimento , Especificidade da EspécieRESUMO
Arabidopsis Fused kinase TWO-IN-ONE (TIO) controls phragmoplast expansion through its interaction with the Kinesin-12 subfamily proteins that anchor the plus ends of interdigitating microtubules in the phragmoplast midzone. Previous analyses of loss-of-function mutants and RNA interference lines revealed that TIO positively controls both somatic and gametophytic cell cytokinesis; however, knowledge of the full spectrum of TIO functions during plant development remains incomplete. To characterize TIO functions further, we expressed TIO and a range of TIO variants under control of the TIO promoter in wild-type Arabidopsis plants. We discovered that TIO-overexpressing transgenic lines produce enlarged pollen grains, arising from incomplete cytokinesis during male meiosis, and show sporophytic abnormalities indicative of polyploidy. These phenotypes arose independently in TIO variants in which either gametophytic function or the ability of TIO to interact with Kinesin-12 subfamily proteins was abolished. Interaction assays in yeast showed TIO to bind to the AtNACK2/TETRASPORE, and plants doubly homozygous for kinesin-12a and kinesin-12b knockout mutations to produce enlarged pollen grains. Our results show TIO to dominantly inhibit male meiotic cytokinesis in a dosage-dependent manner that may involve direct binding to a component of the canonical NACK-PQR cytokinesis signaling pathway.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Citocinese/genética , Regulação da Expressão Gênica de Plantas , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Genes Dominantes/genética , Cinesinas/genética , Cinesinas/metabolismo , Meiose/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Mutagênese Insercional , Fenótipo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Pólen/citologia , Pólen/enzimologia , Pólen/genética , Pólen/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
The micronucleus (MN) assay, applied in different surrogate tissues, is one of the best validated cytogenetic techniques for evaluating chromosomal damage in humans. The cytokinesis-block micronucleus cytome assay (CBMNcyt) in peripheral blood lymphocytes is the most frequent method in biomonitoring human populations to evaluate exposure to genotoxic agents, micronutrient deficiency, or excess and genetic instability. Furthermore recent scientific evidence suggests an association between an increased MN frequency in lymphocytes and risk of cancer and other age-related degenerative diseases. The micronucleus cytome assay applied in buccal exfoliated cells (BMNCyt) provides a complementary method for measuring DNA damage and cytotoxic effects in an easily accessible tissue not requiring in vitro culture. The protocol for CBMNcyt described here refers to the use of ex vivo whole blood involving 72 h of culture with the block of cytokinesis at 44 h. BMNCyt protocol reports the established method for sample processing, slide preparation, and scoring.
Assuntos
Linfócitos/citologia , Linfócitos/metabolismo , Testes para Micronúcleos/métodos , Mucosa Bucal/citologia , Mucosa Bucal/metabolismo , Coleta de Amostras Sanguíneas , Separação Celular , Citocinese/genética , HumanosRESUMO
The microtubule (MT)-associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss-of-function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N-terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild-type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen-activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast-based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Citocinese/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Endosperma/genética , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Houttuynia cordata (Saururaceae) is a leaf vegetable and a medicinal herb througout much of Asia. Cytomixis and meiotic abnormalities during microsporogenesis were found in two populations of H. cordata with different ploidy levels (2n = 38, 96). Cytomixis occurred in pollen mother cells during meiosis at high frequencies and with variable degrees of chromatin/chromosome transfer. Meiotic abnormalities, such as chromosome laggards, asymmetric segregation and polyads, also prevailed in pollen mother cells at metaphase of the first division and later stages. They were caused by cytomixis and resulted in very low pollen viability and male sterility. Pollen mother cells from the population with 2n = 38 showed only simultaneous cytokinesis, but most pollen mother cells from the population with 2n = 96 showed successive cytokinesis; a minority underwent simultaneous cytokinesis. Cytomixis and irregular meiotic divisions appear to be the origin of the intraspecific polyploidy in this species, which has large variations in chromosome numbers.
Assuntos
Aberrações Cromossômicas , Cromossomos de Plantas/genética , Gametogênese Vegetal/genética , Variação Genética , Houttuynia/genética , Infertilidade das Plantas , Cromatina/genética , Segregação de Cromossomos/genética , Citocinese/genética , Meiose/genética , Plantas Medicinais/genética , Ploidias , Pólen/genéticaRESUMO
BACKGROUND: The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. RESULTS: We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. CONCLUSION: Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/fisiologia , Citocinese/efeitos dos fármacos , Dinaminas/antagonistas & inibidores , Genes bcl-2/fisiologia , Neoplasias/patologia , Apoptose/genética , Apoptose/fisiologia , Caspases/metabolismo , Linhagem Celular Tumoral , Citocinese/genética , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HT29 , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Transfecção , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
Several mutations are known to alter the normal progression of meiosis and can be correlated with defects in microtubule distribution. The dv mutation affects the spindle organization and chromosomes do not converge into focused poles. Two Brachiaria hybrids presented the phenotypic expressions of dv mutation but exhibited many more details in the second division. Bivalents were distantly positioned and spread over a large metaphase plate and failed to converge into focused poles. Depending on the distance of chromosomes at the poles, telophase I nuclei were elongated or the chromosomes were grouped into various micronuclei of different sizes in each cell. The first cytokinesis occurred. However, when there were micronuclei, a second cytokinesis immediately took place dividing the prophase II meiocytes into three or four cells. In each meiocyte, meiosis progressed to the second division. Slightly elongated nuclei or micronuclei were recorded in telophase II. After a third cytokinesis, hexads or octads were formed. Pollen grains of different sizes were generated. One of these hybrids presented a higher frequency of abnormal cells than when previously analyzed. The fate of these hybrids as genitors or as candidates for cultivars in the Brachiaria breeding program is discussed.
Assuntos
Brachiaria/genética , Quimera/genética , Regulação da Expressão Gênica de Plantas/genética , Meiose/genética , Mutação/genética , Fuso Acromático/genética , Brachiaria/crescimento & desenvolvimento , Brachiaria/metabolismo , Ciclo Celular/genética , Segregação de Cromossomos/genética , Citocinese/genética , Micronúcleo Germinativo/genética , Fenótipo , Pólen/citologia , Pólen/genética , Reprodução/genética , Telófase/genéticaRESUMO
Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing approximately 75%, approximately 50%, and approximately 25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation.