No description available.

The multilayered structure (MLS), a component of the locomotory complex of plant sperm, has been utilized extensively by taxonomists in establishing phylogenetic relationships between the lower plants and algae. Unfortunately, there has been almost no biochemical characterization of the MLS and, in those studies that did attempt a characterization, conflicting results were obtained. We utilized antisera to the calcium-binding protein centrin to probe thin sections of the mid-stage spermatids of the anthocerote Phaeoceros laevis, the hepatic Sphaerocarpos texanus, and the pteridophyte Ceratopteris richardii embedded in L. R. White resin. The lamellar strip (LS; layers S2-S4) of the MLS in each of these species is labelled strongly with anti-centrin, but the S1 layer, composed of microtubules, is not. In Ceratopteris, centrin is also detected in the amorphous electron opaque material that connects the basal bodies of the flagella. Both the MLS and the amorphous zones are putative microtubule organizing centers. Extracts from axenic cultures of Ceratopteris subjected for electrophoresis and Western blotting revealed a reactive band at 19.3 kDa, a protein similar in molecular mass to algal centrin. These data indicate that the MLS is composed at least partially of the protein centrin or a protein antigenically-related to centrin. This report is the first electron microscopic immunocytochemical demonstration that a centrin homologue is found in land plants and that it occurs at putative microtubule organizing centers.

Blastocladiella emersonii é um fungo aquático da classe dos quitridiomicetos, notável pelas mudanças morfogenéticas que ocorrem durante o seu ciclo de vida. Neste trabalho isolamos 8.495 seqüências expressas (ESTs) deste fungo, que representam 3.226 seqüências únicas putativas. Destas seqüências, 37% foram classificadas segundo o processo biológico onde estariam envolvidas, de acordo com o sistema de anotação do Gene Ontology (GO). Analisamos os perfis de expressão in silico das ESTs usando estatística Bayesiana e os resultados obtidos foram validados por Northern blot para sete perfis de expressão selecionados. Pudemos encontrar boa correlação entre vários padrões de expressão e determinados processos biológicos. Foram selecionadas algumas seqüências potencialmente envolvidas com a esporulação do fungo para melhor caracterização. Analisamos a expressão de dois genes codificando centrinas (BeCenl e BeCen2) pertencentes a subfamílias distintas. Centrinas são proteínas ligantes de cálcio envolvidas em diferentes processos como o direcionamento do aparelho flagelar e a duplicação dos centros organizadores de microtúbulos (MTOCs). Observamos que os níveis da proteína BeCenl, que não havia sido descrita em fungos, apresentam um máximo aos 150 min da esporulação, defasado do pico de expressão do seu mRNA que ocorre aos 90 min deste estágio. A proteína BeCen2 está presente em níveis constantes durante todo a ciclo de vida do fungo, embora o seu mRNA apresente um pico de expressão aos 120 min da esporulação. Experimentos de imunofluorescência localizaram a proteína BeCenl no citoplasma e no corpo basal do zoósporo. Estes dados sugerem que BeCenl atue na re-orientação e movimento dos corpos basais e BeCen2 na duplicação dos MTOCs. Investigamos também a expressão de dois genes codificando proteína-quinases dependentes de ciclina putativas (BeCdkl e BeCdk2). Apenas uma Cdk (Cdkl) foi descrita em fungos como diretamente envolvida no controle do ciclo celular. Ambos os genes apresentam expressão diferencial, com níveis máximos de mRNA para os dois casos aos 90 min da esporulação. Por outro lado, a proteína BeCdkl está presente durante todo o ciclo de vida do fungo e foi localizada no núcleo e no capacete nuclear dos zoósporos. Um transportador putativo de hexose (Bemst) foi também analisado, com base na regulação por glicose de genes envolvidos com o ciclo celular observada em eucariotos. Verificou-se que os níveis do mRNA de Bemst diminuem drasticamente durante a esporulação, mas glicose ou outras hexoses não afetaram a expressão de Bemst. / Blastocladiella emersonii is an aquatic fungus that belongs to the class of chytridiomycetes, notable for the morphogenetic processes which occur during its life cycle. In this work we have isolated 8,495 expressed sequence tags (ESTs) from this fungus, representing 3,226 putative unigenes. From these unigenes, 37% were classified into a biological process, as a result of Gene Ontology (GO) annotation. Furthermore, we analyzed the expression profile in silico of each transcript using Bayesian Statistics and seven of these profiles were validated by Northern blot analysis. In addition, we found a good correlation between several of these expression patterns and certain biological processes. Some ESTs potentially involved in the sporulation of the fungus were selected to be further characterized. We analyzed the expression of two genes encoding two centrins (BeCenl and BeCen2) of distinct subfamilies. Centrins are calcium-binding proteins involved in different processes such as basal body orientation and duplication of the microtubuleorganizing centers (MTOCs). The amount of BeCenl, a centrin ortholog not previously described in fungi, presents a maximum at 150 min of sporulation, whereas the peak of its mRNA occurs at 90 min of this stage. Protein BeCen2 presents constant levels during the entire life cycle of the fungus, even though its mRNA shows a peak of expression at 120 min of sporulation. In addition, immunofluorescent studies localized BeCenl in the cytoplasm and the basal body of zoospores. These results suggest that BeCenl plays a role in re-orientation and movement of basal bodies and BeCen2 in MTOCs duplication. We also investigated the expression of two genes encoding putative cyclin-dependent protein kinases (BeCdkl and BeCdk2). Only one type of Cdk (Cdkl) directly involved in cell cycle control has been described in other fungi. Both genes were found to be differentially expressed, with maximum mRNA levels being detected in either case at 90 min of sporulation. In contrast, BeCdk1 is present throughout the life cycle of the fungus and was immunolocalized in the nuc1eus and the nuclear cap of zoospores. A putative hexose transporter (Bemst) was also investigated, taking into account the regulation by glucose of cell cycle controlled genes in eukaryotes. We found that Bemst mRNA levels decrease drastically during sporulation, but glucose and other hexoses had no effect on Bemst expression.

Blastocladiella emersonii é um fungo aquático da classe dos quitridiomicetos, notável pelas mudanças morfogenéticas que ocorrem durante o seu ciclo de vida. Neste trabalho isolamos 8.495 seqüências expressas (ESTs) deste fungo, que representam 3.226 seqüências únicas putativas. Destas seqüências, 37% foram classificadas segundo o processo biológico onde estariam envolvidas, de acordo com o sistema de anotação do Gene Ontology (GO). Analisamos os perfis de expressão in silico das ESTs usando estatística Bayesiana e os resultados obtidos foram validados por Northern blot para sete perfis de expressão selecionados. Pudemos encontrar boa correlação entre vários padrões de expressão e determinados processos biológicos. Foram selecionadas algumas seqüências potencialmente envolvidas com a esporulação do fungo para melhor caracterização. Analisamos a expressão de dois genes codificando centrinas (BeCenl e BeCen2) pertencentes a subfamílias distintas. Centrinas são proteínas ligantes de cálcio envolvidas em diferentes processos como o direcionamento do aparelho flagelar e a duplicação dos centros organizadores de microtúbulos (MTOCs). Observamos que os níveis da proteína BeCenl, que não havia sido descrita em fungos, apresentam um máximo aos 150 min da esporulação, defasado do pico de expressão do seu mRNA que ocorre aos 90 min deste estágio. A proteína BeCen2 está presente em níveis constantes durante todo a ciclo de vida do fungo, embora o seu mRNA apresente um pico de expressão aos 120 min da esporulação. Experimentos de imunofluorescência localizaram a proteína BeCenl no citoplasma e no corpo basal do zoósporo. Estes dados sugerem que BeCenl atue na re-orientação e movimento dos corpos basais e BeCen2 na duplicação dos MTOCs. Investigamos também a expressão de dois genes codificando proteína-quinases dependentes de ciclina putativas (BeCdkl e BeCdk2). Apenas uma Cdk (Cdkl) foi descrita em fungos como diretamente envolvida no controle do ciclo celular. Ambos os genes apresentam expressão diferencial, com níveis máximos de mRNA para os dois casos aos 90 min da esporulação. Por outro lado, a proteína BeCdkl está presente durante todo o ciclo de vida do fungo e foi localizada no núcleo e no capacete nuclear dos zoósporos. Um transportador putativo de hexose (Bemst) foi também analisado, com base na regulação por glicose de genes envolvidos com o ciclo celular observada em eucariotos. Verificou-se que os níveis do mRNA de Bemst diminuem drasticamente durante a esporulação, mas glicose ou outras hexoses não afetaram a expressão de Bemst. / Blastocladiella emersonii is an aquatic fungus that belongs to the class of chytridiomycetes, notable for the morphogenetic processes which occur during its life cycle. In this work we have isolated 8,495 expressed sequence tags (ESTs) from this fungus, representing 3,226 putative unigenes. From these unigenes, 37% were classified into a biological process, as a result of Gene Ontology (GO) annotation. Furthermore, we analyzed the expression profile in silico of each transcript using Bayesian Statistics and seven of these profiles were validated by Northern blot analysis. In addition, we found a good correlation between several of these expression patterns and certain biological processes. Some ESTs potentially involved in the sporulation of the fungus were selected to be further characterized. We analyzed the expression of two genes encoding two centrins (BeCenl and BeCen2) of distinct subfamilies. Centrins are calcium-binding proteins involved in different processes such as basal body orientation and duplication of the microtubuleorganizing centers (MTOCs). The amount of BeCenl, a centrin ortholog not previously described in fungi, presents a maximum at 150 min of sporulation, whereas the peak of its mRNA occurs at 90 min of this stage. Protein BeCen2 presents constant levels during the entire life cycle of the fungus, even though its mRNA shows a peak of expression at 120 min of sporulation. In addition, immunofluorescent studies localized BeCenl in the cytoplasm and the basal body of zoospores. These results suggest that BeCenl plays a role in re-orientation and movement of basal bodies and BeCen2 in MTOCs duplication. We also investigated the expression of two genes encoding putative cyclin-dependent protein kinases (BeCdkl and BeCdk2). Only one type of Cdk (Cdkl) directly involved in cell cycle control has been described in other fungi. Both genes were found to be differentially expressed, with maximum mRNA levels being detected in either case at 90 min of sporulation. In contrast, BeCdk1 is present throughout the life cycle of the fungus and was immunolocalized in the nuc1eus and the nuclear cap of zoospores. A putative hexose transporter (Bemst) was also investigated, taking into account the regulation by glucose of cell cycle controlled genes in eukaryotes. We found that Bemst mRNA levels decrease drastically during sporulation, but glucose and other hexoses had no effect on Bemst expression.

Le centrosome est le centre organisateur des microtubules dans les cellules animales, il nucléé les microtubules interphasiques ainsi que le fuseau mitotique. Les centrosomes sont produits par duplication, mécanisme rigoureusement régulé au cours du cycle cellulaire. En effet, un centrosome comporte deux centrioles qui se dupliquent une fois par cycle cellulaire. Des erreurs de duplication conduisant à plus de deux centrosomes induisent la formation de fuseaux multipolaires et provoquent des défauts de ségrégation des chromosomes. Chez la levure Schizosaccharomyces pombe, un organisme modèle pour l’étude de la division cellulaire, les homologues des centrosomes sont les SPBs (pour Spindle Pole Body). Une structure annexe spécifique liée aux SPBs est appelée demi-pont (quand les SPBs ne sont pas dupliqués) puis pont (quand elle relie les deux SPBs dupliqués). Les deux principaux composants du pont chez la levure S. pombe sont Cdc31 et Sfi1. Sfi1 est une protéine linéaire formée de répétitions en hélice α formant des sites de liaison pour la Centrine/Cdc31. Sfi1 s’assemble en réseau de molécules parallèles interagissant avec le SPB via leur domaine N-terminal. Lors de la première partie de ma thèse, j’ai démontré que Sfi1 est requis pour la duplication et la séparation des deux SPBs. Dans la première partie de ma thèse, je me suis intéressée aux fonctions de Sfi1 chez la levure. Cette étude a permis de démontrer que Sfi1 est un composant du demi-pont et qu’il est essentiel pour la duplication des SPBs et l’assemblage d’un fuseau bipolaire. De plus, nous avons déterminé que le pont est dupliqué en fin de mitose. Enfin, nous avons aussi montré que la déstabilisation du pont menant à sa rupture en mitose, dépend de la phosphorylation de Cdc31 par la kinase mitotique Cdk1. Lors de la seconde partie de ma thèse, je me suis intéressée au complexe Sfi1/Centrine dans les cellules humaines. J’ai confirmé que Sfi1 est localisée aux centrioles. De plus, j’ai montré que la déplétion de Sfi1 dans les cellules RPE1, conduit à une perte de localisation de la Centrine, suggérant soit un défaut de recrutement, soit une déstabilisation. De plus, en absence de Sfi1, les cellules RPE1 ne sont plus capables de former de cil primaire. Ce résultat suggère que Sfi1 et la Centrine sont requis pour la ciliogénèse. Enfin, j’ai aussi démontré que la déplétion deSfi1 induit un arrêt de cycle cellulaire dans les cellules non tumorales RPE1. Dans les cellules cancéreuses, HeLa, le cycle n’est pas arrêté mais j’ai pu observer une prolongation du temps de mitose. En conclusion mes travaux montrent que bien que la fonction de Sfi1/Centrin ne soit pas conservée, le complexe reste essentiel pour l’intégrité structurale et fonctionnelle du centrosome. / The centrosome is the main microtubule organizing center. It nucleates and organizes interphase microtubule and contributes to the assembly of the bipolar mitotic spindle. To do so, the centrosome, present in one copy at the beginning of the cell cycle, duplicates to produce a second copy. The duplication process is tightly controlled and regulated since centrosome over-duplication can lead to multipolar mitotic spindles and promote genome instability and tumorigenesis. The duplication of the yeast centrosome, the SPB (Spindle pole body), begins with the duplication of the half bridge. This appendage is composed of Sfi1/Cdc31 complex organized in a parallel array attached to the core SPB. SPB duplication relies on the assembly of a second array of Sfi1/Cdc31, anti-parallel to the first one, creating thereby an assembly site for the new SPB. Therefore Sfi1 is essential for SPB duplication and our work defined the timing of half-bridge duplication and some of the regulatory mechanisms that favor bridge splitting to release duplicated centrosomes and allow spindle assembly at mitotic onset. Sfi1 and Cdc31/Centrins are conserved in human cells where the centrosome is composed of two centrioles surrounded by the pericentriolar material. Centrins are concentrated in the distal end of centrioles. Sfi1 has also been localized to centrioles, but its function remained unknown. Thus, we started investigating Sfi1 function in human cells. We found that Sfi1 depletion leads to a decrease in Centrin recruitment to the centrioles. It also leads to a cell cycle arrest in G1 in RPE1 cells, an event previously observed in presence of defects in centriole biogenesis. In HeLa cells where the cell cycle is not affected, Sfi1 depletion leads to a mitotic delay. Moreover, Sfi1 depletion leads to cilium assembly. To conclude, these results altogether point towards a role of human Sfi1 in centriole biogenesis.

No description available.