Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89.: Activation of Aurora-A by Nucleophosmin

International audienceAurora 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

Implications des harpines HrpNea et HrpWea, protéines de pathogénie de la bactérie Erwinia amylovora dans les étapes précoces d'interaction avec la cellule végétale

Les harpines sont des effecteurs protéiques sécrétés par le système de sécrétion de type III de certaines bactéries phytopathogènes. L'objectif des travaux présentés dans cette thèse fut d'étudier l'implication des harpines HrpNea et HrpWea dans l'interaction entre Erwinia amylovora et la cellule végétale. Dans un premier temps, nous avons travaillé avec des cellules en suspension d'Arabidopsis thaliana, plante non-hôte d'E. amylovora (situation incompatible). Sur ce modèle, l'inhibition des canaux anioniques est un événement précoce déterminant conduisant à la mort cellulaire en réponse à HrpNea. Nous nous sommes ensuite intéressés aux effets de HrpWea, l'autre harpine d'E. amylovora. Nos résultats montrent que HrpWea est une harpine dont les effets sur la mort cellulaire sont doubles. A 200 nM, HrpWea induit la mort tandis qu'à 0,2 nM, HrpWea est capable d'inhiber la mort cellulaire induite par la harpine HrpNea. La modulation des canaux anioniques apparaît aussi comme un événement déterminant de l'effet " protecteur " de HrpWea. Plus largement, ces résultats suggèrent que HrpWea peut être considérée comme un inhibiteur des réactions de défense de la cellule végétale. Enfin, nous avons étudié les effets de HrpNea chez un modèle cellulaire hôte (situation compatible), les cellules en suspension de pommier. Nous avons montré que les réponses induites par HrpNea en contexte hôte et en contexte non-hôte sont différentes, ce qui explique vraisemblablement le fait qu'HrpNea n'induit pas la mort chez ses plantes hôtes.Considérées dans leur ensemble, ces données ont permis d'améliorer la compréhension de l'implication des harpines d'E. amylovora dans la pathogénie et la réponse hypersensible induites par cette bactérie. D'une façon plus générale, ils ont aussi permis d'apporter de nouvelles informations quant au rôle des canaux ioniques dans les phénomènes de mort cellulaire programmée.Harpins are protean effectors secreted by type III secretion system of phytopathogenic bacteria. The aim of the present work was to investigate the involvement of HrpNea and HrpWea harpins from Erwinia amylovora during the interaction between this bacteria and the plant cell. We first worked with suspension cells from the non-host plant Arabidopsis thaliana (incompatible situation). On this model, we showed that anion channel inhibition is a determinant event leading to cell death triggered by HrpNea. We then studied the effects of HrpWea. Our results showed that HrpWea has different effects depending on its concentration. At high concentration , HrpWea induced cell death, whereas at low concentration , HrpWea was able to inhibit HrpNea induced cell death. Anion channel modulation also appeared to be a determinant event of cell death inhibition. More widely, these results suggested that HrpWea could be a plant cell defense mechanism inhibitor. Finally, we studied the effects of HrpNea on the host apple cells (compatible situation). We showed differential responses between host and non-host plant cells, likely explaining that HrpNea did not trigger cell death in host plants.Taken together, these data allowed us to improve our knowledge concerning involvement of HrpNea and HrpWea in E. amylovora hypersensitive response and pathogenicity. Moreover, these results bring new data concerning the role of ion channels during cell death.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Aurora A's Functions During Mitotic Exit: The Guess Who Game

International audienceUntil 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 mechanism

Ion Transport in Plant Cell Shrinkage During Death

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THE SPLICING FACTOR PTBP1 REPRESSES TP63 γ ISOFORM PRODUCTION IN SQUAMOUS CELL CARCINOMA

ABSTRACT The TP63 gene encodes the transcription factor p63. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, β, γ and δ. The regulatory functions of p63 may be isoform-specific. The α isoform inhibits the epithelial to mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Here, we observed in TCGA data that a high ratio of the TP63γ isoform to the other isoforms is a pejorative factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC). We therefore addressed the regulation of the γ isoform. In several tissues (GTEX data), the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ . Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines leads to an increase in abundance of the γ isoform. 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. The region around the TP63γ -specific exon was sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Finally, we demonstrated that the regulation of TP63γ production by PTBP1 is conserved in amphibians, revealing that it encounters a strong evolutionary pressure. Together, these results identify TP63γ as a prognostic marker in HNSCC, and identify PTBP1 as a direct negative regulator of its production

Cryptogein-induced anion effluxes: electrophysiological properties and analysis of the mechanisms through which they contribute to the elicitor-triggered cell death

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Cryptogein-induced anion effluxes: electrophysiological properties and analysis of the mechanisms through which they contribute to the elicitor-triggered cell death

International audienceAnion 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 (NO3−) efflux. This efflux is an early prerequisite to the cryptogein‑triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor‑mediated NO3− 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 Ca2+ influx, an essential component of elicitor signalling for HR cell death, were prevented by inhibiting the NO3− efflux. Similarly, pharmacological blocking of the anion efflux suppressed vacuolar collapse, a hallmark of cell death. The role of NO3− 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 NO3− 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

Cryptogein-Induced Anion Effluxes: Electrophysiological Properties and Analysis of the Mechanisms Through Which They Contribute to the Elicitor-Triggered Cell Death

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 (NO3−) efflux. This efflux is an early prerequisite to the cryptogein-triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor-mediated NO3− 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 Ca2+ influx, an essential component of elicitor signalling for HR cell death, were prevented by inhibiting the NO3− efflux. Similarly, pharmacological blocking of the anion efflux suppressed vacuolar collapse, a hallmark of cell death. The role of NO3− 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 NO3− 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

The Splicing Factor PTBP1 Represses TP63 γ Isoform Production in Squamous Cell Carcinoma

International audienceThe 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, β, 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