RESUMO
CD44 mRNA contains nine consecutive cassette exons, v2 to v10. Upon alternative splicing, several isoforms are produced with different impacts on tumor biology. Here, we demonstrate the involvement of the RNA-binding proteins CELF1 and ELAVL1 in the control of CD44 splicing. We show by FRET-FLIM that these proteins directly interact in the nucleus. By combining RNAi-mediated depletion and exon array hybridization in HeLa cells, we observe that the exons v7 to v10 of CD44 are highly sensitive to CELF1 and ELAVL1 depletion. We confirm by RT-PCR that CELF1 and ELAVL1 together stimulate the inclusion of these exons in CD44 mRNA. Finally, we show in eight different tumor types that high expression of CELF1 and/or ELAVL1 is correlated with the inclusion of CD44 variable exons. These data point to functional interactions between CELF1 and ELAVL1 in the control of CD44 splicing in human cancers.
Assuntos
Processamento Alternativo , Receptores de Hialuronatos , Proteínas CELF1 , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Éxons/genética , Células HeLa , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
During mitosis, the cell sequentially constructs two microtubule-based spindles to ensure faithful segregation of chromosomes. A bipolar spindle first pulls apart the sister chromatids, then a central spindle further separates them away. Although the assembly of the first spindle is well described, the assembly of the second remains poorly understood. We report here that the inhibition of Aurora A leads to an absence of the central spindle resulting from a lack of nucleation of microtubules in the midzone. In the absence of Aurora A, the HURP (also known as DLGAP5) and NEDD1 proteins that are involved in nucleation of microtubules fail to concentrate in the midzone. HURP is an effector of RanGTP, whereas NEDD1 serves as an anchor for the γ-tubulin ring complex (γTURC). Interestingly, Aurora A phosphorylates HURP and NEDD1 during assembly of the initial bipolar spindle. We show here that the expression of a NEDD1 isoform mimicking phosphorylation by Aurora A is sufficient to restore microtubule nucleation in the midzone under conditions of Aurora A inhibition. These results reveal a new control mechanism of microtubule nucleation by Aurora A during assembly of the central spindle.
Assuntos
Aurora Quinase A/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Anáfase/fisiologia , Aurora Quinase A/antagonistas & inibidores , Linhagem Celular Tumoral , Citocinese/fisiologia , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Fosforilação , Serina/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: Ocular lens clouding is termed as cataract, which depending on the onset, is classified as congenital or age-related. Developing new cataract treatments requires new models. Thus far, Xenopus embryos have not been evaluated as a system for studying cataract. RESULTS: We characterized the developmental process of lens formation in Xenopus laevis tailbuds and tadpoles, and we disrupted the orthologues of three mammalian cataract-linked genes in F0 by CRISPR/Cas9. We assessed the consequences of gene inactivation by combining external examination with histochemical analyses and functional vision assays. Inactivating the key metazoan eye development transcription factor gene pax6 produces a strong eye phenotype including an absence of eye tissue. Inactivating the genes for gap-junction protein and a nuclease, gja8 and dnase2b, produces lens defects that share several features of human cataracts, including impaired vision acuity, nuclei retention in lens fiber cells, and actin fibers disorganization. We tested the potential improvement of the visual acuity of gja8 crispant tadpoles upon treatment with the molecular chaperone 4-phenylbutyrate. CONCLUSION: Xenopus is a valuable model organism to understand the molecular pathology of congenital eye defects, including cataracts, and to screen molecules with a potential to prevent or reverse cataracts.
Assuntos
Xenopus laevis/fisiologia , Animais , Catarata/fisiopatologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Cristalino/fisiologiaRESUMO
During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper.
Assuntos
Aurora Quinase A/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Mad2/genética , Prometáfase/genética , Anáfase/genética , Aurora Quinase A/antagonistas & inibidores , Azepinas/farmacologia , Linhagem Celular Tumoral , Cromátides/genética , Segregação de Cromossomos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Cinetocoros/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Mitose/genética , Nocodazol/farmacologia , Paclitaxel/farmacologia , Prometáfase/efeitos dos fármacos , Pirimidinas/farmacologia , Fuso Acromático/genéticaRESUMO
Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium- and phospholipid-binding protein implicated in cortical actin remodeling. We report that annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In the absence of annexin A2, the small GTPase RhoA-which regulates cortical cytoskeletal rearrangement-fails to form a compact ring at the equatorial plane. Furthermore, annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in central spindle-equatorial plasma membrane communication.
Assuntos
Anexina A2/genética , Citocinese/genética , Osteoblastos/metabolismo , Fuso Acromático/metabolismo , Anexina A2/antagonistas & inibidores , Anexina A2/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cromátides/metabolismo , Cromátides/ultraestrutura , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Osteoblastos/ultraestrutura , Mutação Puntual , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Transdução de Sinais , Fuso Acromático/ultraestrutura , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína Vermelha FluorescenteRESUMO
Cataract, the opacification of the lens, is the leading cause of blindness worldwide. Although effective, cataract surgery is costly and can lead to complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined three-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization and biology. These organoids can be rapidly produced in large amounts. High-throughput RNA sequencing (RNA-seq) on specific organoid regions isolated via laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display a spatiotemporal expression of key lens genes, e.g., Jag1, Pax6, Prox1, Hsf4 and Cryab. Further, these lens organoids are amenable to the induction of opacities. Finally, the knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1, induces opacities in these organoids, indicating their use in rapidly screening for genes that are functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataracts.
Assuntos
Catarata , Cristalino , Animais , Camundongos , Cristalino/metabolismo , Catarata/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a RNA/metabolismo , Organoides/metabolismoRESUMO
The ocular lens, along with the cornea, focuses light on the retina to generate sharp images. Opacification of the lens, or cataract, is the leading cause of blindness worldwide. Presently, the best approach for cataract treatment is to surgically remove the diseased lens and replace it with an artificial implant. Although effective, this is costly and can have post-surgical complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and anti-cataract drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined 3-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization at the tissue, cellular and transcriptomic levels. These 3D cultured lens organoids can be rapidly produced in large amounts. High-throughput RNA-sequencing (RNA-seq) on specific organoid regions isolated by laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display spatiotemporal expression of key lens genes, e.g. , Jag1 , Pax6 , Prox1 , Hsf4 and Cryab . Further, these lens organoids are amenable to induction of opacities. Finally, knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1 , induces opacities in these organoids, indicating their use in rapidly screening for genes functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology, and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataract.
RESUMO
The TP63 gene encodes the p63 transcription factor. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, ß, γ, and δ. The regulatory functions of p63 are isoform specific. The α isoform inhibits the epithelial-to-mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Using The Cancer Genome Atlas data, we observed that a higher proportion of the TP63γ isoform is a detrimental factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC) and is accompanied by the downregulation of desmosomal genes. By a correlation-based approach, we investigated the regulation of the production of the TP63γ isoform. According to our analysis of GTEx data, the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ in several tissues. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines, keratinocyte or Xenopus embryos leads to an increase in TP63γ isoform abundance. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. Intronic regions around the TP63γ-specific exon were sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Together, these results identify TP63γ as an unfavorable prognostic marker in HNSCC, and identify PTBP1 as the first direct splicing regulator of TP63γ production and a potential route toward TP63 isoform control. Significance: Quantifying TP63γ isoforms in patients' tumors could allow for the early detection of patients with HNSCC with an early loss in desmosomal gene expression and poor prognostic. The identification of PTBP1 as a transacting factor controlling TP63γ production may allow to control TP63γ expression.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Humanos , Fatores de Processamento de RNA/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Isoformas de Proteínas/genética , Processamento Alternativo/genética , Fatores de Transcrição/genética , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Proteínas Supressoras de Tumor/genética , Ribonucleoproteínas Nucleares Heterogêneas/genéticaRESUMO
Hyperosmotic stresses represent some of the most serious abiotic factors that adversely affect plants growth, development and fitness. Despite their central role, the early cellular events that lead to plant adaptive responses remain largely unknown. In this study, using Arabidopsis thaliana cultured cells we analyzed early cellular responses to sorbitol-induced hyperosmotic stress. We observed biphasic and dual responses of A. thaliana cultured cells to sorbitol-induced hyperosmotic stress. A first set of events, namely singlet oxygen (1O2) production and cell hyperpolarization due to a decrease in anion channel activity could participate to signaling and osmotic adjustment allowing cell adaptation and survival. A second set of events, namely superoxide anion (O2-) production by RBOHD-NADPH-oxidases and SLAC1 anion channel activation could participate in programmed cell death (PCD) of a part of the cell population. This set of events raises the question of how a survival pathway and a death pathway could be induced by the same hyperosmotic condition and what could be the meaning of the induction of two different behaviors in response to hyperosmotic stress.
Assuntos
Apoptose/efeitos dos fármacos , Arabidopsis/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Osmorregulação/efeitos dos fármacos , Pressão Osmótica/efeitos dos fármacos , Sorbitol/metabolismoRESUMO
Erwinia amylovora is a gram-negative necrogenic bacterium causing fire blight of the Maloideae subfamily of Rosaceae such as apple and pear. It provokes progressive necrosis in aerial parts of susceptible host plants (compatible interaction) and a hypersensitive reaction (HR) when infiltrated in nonhost plants (incompatible interaction). The HrpN(ea) harpin is a type three secretion system effector secreted by E. amylovora. This protein is involved in pathogenicity and HR-eliciting capacity of E. amylovora. In the present study, we showed that, in nonhost Arabidopsis thaliana cells, purified HrpN(ea) induces cell death and H2O2 production, two nonhost resistance responses, but failed to induce such responses in host MM106 apple cells. Moreover, HrpN(ea) induced an increase in anion current in host MM106 apple cells, at the opposite of the decrease of anion current previously shown to be necessary to induce cell death in nonhost A. thaliana cells. These results suggest that HrpN(ea) induced different signaling pathways, which could account for early induced compatible or incompatible interaction development.
Assuntos
Arabidopsis/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/farmacologia , Erwinia amylovora/metabolismo , Malus/efeitos dos fármacos , Arabidopsis/citologia , Arabidopsis/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletrofisiologia , Erwinia amylovora/genética , Erwinia amylovora/patogenicidade , Malus/citologia , Malus/microbiologia , Praguicidas/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologiaRESUMO
Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation.
Assuntos
Aurora Quinase A/metabolismo , Técnicas Biossensoriais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Erwinia amylovora is a necrogenic bacterium that causes fire blight of the Maloideae subfamily of Roseacae, such as apple and pear. It provokes necrosis in aerial parts of susceptible host plants and the typical hypersensitive reaction in non-host plants. The secreted harpin, HrpN ea, is able by itself to induce an active cell death in non-host plants. Ion flux modulations were shown to be involved early in such processes but very few data are available on the plasma membrane ion channel activities responsible for the pathogen-induced ion fluxes. We show here that HrpN ea induces cell death in non-host Arabidopsis thaliana suspension cells. We further show that two cystic fibrosis transmembrane conductance regulator modulators, glibenclamide and bromotetramisole, can regulate anion channel activities and HrpN ea-induced cell death.
Assuntos
Arabidopsis/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Erwinia amylovora/metabolismo , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/farmacologia , Técnicas de Cultura de Células , Morte Celular , Glibureto/farmacologia , Ativação do Canal Iônico , Tetramizol/farmacologiaRESUMO
Until recently, the knowledge of Aurora A kinase functions during mitosis was limited to pre-metaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. However, an involvement of Aurora A in post-metaphase events was also suspected, but not clearly demonstrated due to the technical difficulty to perform the appropriate experiments. Recent developments of both an analog-specific version of Aurora A and small molecule inhibitors have led to the first demonstration that Aurora A is required for the early steps of cytokinesis. As in pre-metaphase, Aurora A plays diverse functions during anaphase, essentially participating in astral microtubules dynamics and central spindle assembly and functioning. The present review describes the experimental systems used to decipher new functions of Aurora A during late mitosis and situate these functions into the context of cytokinesis mechanisms.
RESUMO
Signals leading to mycorrhizal differentiation are largely unknown. We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius. This fungi establishes ectomycorrhizas with Eucalyptus globulus. Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A. thaliana, as previously reported for the host plant. In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents. These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A. thaliana and provide tools for the dissection of the hypaphorine-auxin interaction.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Indóis/farmacologia , Micorrizas/metabolismo , Proteínas de Plantas , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Antagonismo de Drogas , Glutationa Transferase/efeitos dos fármacos , Glutationa Transferase/genética , Indóis/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Micorrizas/crescimento & desenvolvimento , Ácidos Naftalenoacéticos/farmacologia , Proteínas Nucleares/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologiaRESUMO
Knowledge of Aurora A kinase functions is limited to premetaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. The involvement of Aurora A in events after metaphase has only been suggested because appropriate experiments are technically difficult. We report here the design of the first human Aurora A kinase (as-AurA) engineered by chemical genetics techniques. This kinase is fully functional biochemically and in cells, and is rapidly and specifically inhibited by the ATP analogue 1-Naphthyl-PP1 (1-Na-PP1). By treating cells exclusively expressing the as-AurA with 1-Na-PP1, we discovered that Aurora A is required for central spindle assembly in anaphase through phosphorylation of Ser 19 of P150Glued. This paper thus describes a new Aurora A function that takes place after the metaphase-to-anaphase transition and a new powerful tool to search for and study new Aurora A functions.
Assuntos
Anáfase/fisiologia , Metáfase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Anáfase/efeitos dos fármacos , Aurora Quinases , Linhagem Celular , Complexo Dinactina , Humanos , Metáfase/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Serina/genética , Serina/metabolismo , Fuso Acromático/genéticaRESUMO
Aurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA.
Assuntos
Centrossomo/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Aurora Quinases , Células HeLa , Humanos , Proteínas Nucleares/genética , Nucleofosmina , FosforilaçãoRESUMO
We report on the underlying molecular mechanisms likely responsible for the high-level fluconazole resistance in a Candida lusitaniae clinical isolate. Fluconazole resistance correlated with overexpression of ERG11 and of several efflux pump genes, in particular, the orthologs of the Candida albicans MDR1, PDR16, CDR1, CDR2, and YOR1.
Assuntos
Candida/efeitos dos fármacos , Candida/genética , Farmacorresistência Fúngica/genética , Fluconazol/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA , Proteínas Fúngicas/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Metiltransferases/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase/métodosRESUMO
Harpins are type three secretion system (TTSS) effectors. While few harpins are thought to be translocators of TTSS effectors through the host plasma membrane during plant/bacteria interactions, functions of many harpins remain for the moment mysterious. We recently showed that the HrpW(ea) harpin from Erwinia amylovora, at subnamolar concentration, was able to decrease defense responses triggered by another harpin from this bacteria, HrpN(ea). This antagonism could be the result of opposed anion channels modulations triggered by HrpW(ea) and HrpN(ea). At upper concentrations HrpW(ea) alone, or in combination with HrpN(ea), was able to induce cell death. This form of cell death involves strong ion channel activation and shares similarity with apoptosis volume decrease (AVD), a form of programmed cell death well described in animal cells. All these results suggest different ways for harpins to trigger cell death and highlight the role of ion channels during cell death processes.
RESUMO
Anion effluxes are amongst the earliest reactions of plant cells to elicitors of defence responses. However, their properties and their role in disease resistance remain almost unknown. We previously demonstrated that cryptogein, an elicitor of tobacco defence responses, induces a nitrate (NO(3) (-)) efflux. This efflux is an early prerequisite to the cryptogein-triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor-mediated NO(3) (-) efflux and clarified the mechanisms through which it contributes to cell death. Application of the discontinuous single electrode voltage-clamp technique in tobacco cells elicited with cryptogein enabled us to record the activation of slow-type deactivating anion channel currents. Cryptogein-induced plasma membrane depolarization and Ca(2+) influx, an essential component of elicitor signalling for HR cell death, were prevented by inhibiting the NO(3) (-) efflux. Similarly, pharmacological blocking of the anion efflux suppressed vacuolar collapse, a hallmark of cell death. The role of NO(3) (-) efflux in mediating proteases activation was further assessed. It is shown that cryptogein induced the activation of three proteases with apparent molecular masses of 95, 190 and 240 kDa. Their activation occurred independently on the anion efflux and, together with cell death, was strongly reduced by cycloheximide and the protease inhibitor PMSF. In contrast, the NO(3) (-) efflux was shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during the HR. Collectively, our data indicate that anion efflux is an early prerequisite to morphological and biochemical events participating to cell death.