No description available.

La progresión meiótica y el desarrollo embrionario temprano están programados, en parte, por la activación tradcuccional de mRNAs maternos como lo son los que codifican para las proteinas de ciclina B1 o mos. Estos mRNAs no son traducidos al mismo tiempo ni en el mismo lugar. Por lo contrario, su traducción está especificamente regulada por elementos de poliadenilación citoplasmática (CPEs) presentes en sus 3'UTRs. Los elementos CPEs reclutan a la proteina de unión a CPE (CPE-binding protein CPEB (Colegrove-Otero et al., 2005; de Moor et al., 2005; Mendez and Richter, 2001; Richter, 2007)). Esta proteina de unión al RNA no sólo determina cuándo y en qué medida un mRNA será activado traduccionalmente por poliadenilación citoplasmática (Mendez et al., 2000a; Mendez et al., 2000b; Mendez et al., 2002) sino que también participa, junto con el represor de la traducción Maskin, en el transporte y la localización de sus mRNAs diana hacia los sitios de localización subcelular donde su traducción ocurrirá (Huang et al., 2003; Huang and Richter, 2004). Durante el desarrollo embrionario de Xenopus, CPEB se encuentra localizada en el polo animal de los oocitos y más tarde, sobre el huso mitótico y centrosomas en el embrión (Groisman et al., 2000). Se ha demostrado que embriones de Xenopus inyectados con agentes que interrumpen la traducción dependiente de poliadenilación citoplasmática, detienen la división celular y presentan estructuras mitóticas anormales (Groisman et al., 2000). En este trabajo que derivó en mi tesis doctoral, hemos demostrado que la activación traduccional localizada en el huso mitótico de mRNAs regulados por CPEB que codifican para proteinas con una conocida función en aspectos estructurales del ciclo celular como la formación del huso mitótico y la segregación cromosómica, es esencial para completar la primera división meiótica y para la correcta segregación cromosómica en oocitos de Xenopus.

No description available.

Functional characterization of biochemically-isolated proteins is a central task in the biochemical and genetic description of the biology of cells and tissues. Protein identification by mass spectrometry consists of associating an isolated protein with a specific gene or protein sequence in silico, thus inferring its specific biochemical function based upon previous characterizations of that protein or a similar protein having that sequence identity. By performing this analysis on a large scale in conjunction with biochemical experiments, novel biological knowledge can be developed. The study presented here focuses on mass spectrometry-based proteomics of organisms with unsequenced genomes and corresponding developments in biological sequence database searching with mass spectrometry data. Conventional methods to identify proteins by mass spectrometry analysis have employed proteolytic digestion, fragmentation of resultant peptides, and the correlation of acquired tandem mass spectra with database sequences, relying upon exact matching algorithms; i.e. the analyzed peptide had to previously exist in a database in silico to be identified. One existing sequence-similarity protein identification method was applied (MS BLAST, Shevchenko 2001) and one alternative novel method was developed (MultiTag), for searching protein and EST databases, to enable the recognition of proteins that are generally unrecognizable by conventional softwares but share significant sequence similarity with database entries (~60-90%). These techniques and available database sequences enabled the characterization of the Xenopus laevis microtubule-associated proteome and the Dunaliella salina soluble salt-induced proteome, both organisms with unsequenced genomes and minimal database sequence resources. These sequence-similarity methods extended protein identification capabilities by more than two-fold compared to conventional methods, making existing methods virtually superfluous. The proteomics of Dunaliella salina demonstrated the utility of MS BLAST as an indispensable method for characterization of proteins in organisms with unsequenced genomes, and produced insight into Dunaliella?s inherent resilience to high salinity. The Xenopus study was the first proteomics project to simultaneously use all three central methods of representation for peptide tandem mass spectra for protein identification: sequence tags, amino acids sequences, and mass lists; and it is the largest proteomics study in Xenopus laevis yet completed, which indicated a potential relationship between the mitotic spindle of dividing cells and the protein synthesis machinery. At the beginning of these experiments, the identification of proteins was conceptualized as using ?conventional? versus ?sequence-similarity? techniques, but through the course of experiments, a conceptual shift in understanding occurred along with the techniques developed and employed to encompass variations in mass spectrometry instrumentation, alternative mass spectrum representation forms, and the complexities of database resources, producing a more systematic description and utilization of available resources for the characterization of proteomes by mass spectrometry and advanced informatic approaches. The experiments demonstrated that proteomics technologies are only as powerful in the field of biology as the biochemical experiments are precise and meaningful.

No description available.

Functional characterization of biochemically-isolated proteins is a central task in the biochemical and genetic description of the biology of cells and tissues. Protein identification by mass spectrometry consists of associating an isolated protein with a specific gene or protein sequence in silico, thus inferring its specific biochemical function based upon previous characterizations of that protein or a similar protein having that sequence identity. By performing this analysis on a large scale in conjunction with biochemical experiments, novel biological knowledge can be developed. The study presented here focuses on mass spectrometry-based proteomics of organisms with unsequenced genomes and corresponding developments in biological sequence database searching with mass spectrometry data. Conventional methods to identify proteins by mass spectrometry analysis have employed proteolytic digestion, fragmentation of resultant peptides, and the correlation of acquired tandem mass spectra with database sequences, relying upon exact matching algorithms; i.e. the analyzed peptide had to previously exist in a database in silico to be identified. One existing sequence-similarity protein identification method was applied (MS BLAST, Shevchenko 2001) and one alternative novel method was developed (MultiTag), for searching protein and EST databases, to enable the recognition of proteins that are generally unrecognizable by conventional softwares but share significant sequence similarity with database entries (~60-90%). These techniques and available database sequences enabled the characterization of the Xenopus laevis microtubule-associated proteome and the Dunaliella salina soluble salt-induced proteome, both organisms with unsequenced genomes and minimal database sequence resources. These sequence-similarity methods extended protein identification capabilities by more than two-fold compared to conventional methods, making existing methods virtually superfluous. The proteomics of Dunaliella salina demonstrated the utility of MS BLAST as an indispensable method for characterization of proteins in organisms with unsequenced genomes, and produced insight into Dunaliella?s inherent resilience to high salinity. The Xenopus study was the first proteomics project to simultaneously use all three central methods of representation for peptide tandem mass spectra for protein identification: sequence tags, amino acids sequences, and mass lists; and it is the largest proteomics study in Xenopus laevis yet completed, which indicated a potential relationship between the mitotic spindle of dividing cells and the protein synthesis machinery. At the beginning of these experiments, the identification of proteins was conceptualized as using ?conventional? versus ?sequence-similarity? techniques, but through the course of experiments, a conceptual shift in understanding occurred along with the techniques developed and employed to encompass variations in mass spectrometry instrumentation, alternative mass spectrum representation forms, and the complexities of database resources, producing a more systematic description and utilization of available resources for the characterization of proteomes by mass spectrometry and advanced informatic approaches. The experiments demonstrated that proteomics technologies are only as powerful in the field of biology as the biochemical experiments are precise and meaningful.

Les microtubules sont de longs polymères cylindriques de la protéine α, β tubuline, utilisés dans les cellules pour construire le cytosquelette, le fuseau mitotique et les axonèmes. Ces polymères creux sont cruciaux pour de nombreuses fonctions cellulaires, y compris le transport intracellulaire et la ségrégation chromosomique pendant la division cellulaire. Au fur et à mesure que les cellules se développent, se divisent et se différencient, les microtubules passent par un processus, appelé instabilité dynamique, ce qui signifie qu’ils basculent constamment entre les états de croissance et de rétrécissement. Cette caractéristique conservée et fondamentale des microtubules est étroitement régulée par des familles de protéines associées aux microtubules. Les protéines de kinésine-13 sont une famille de facteurs régulateurs de microtubules qui dépolymérisent catalytiquement les extrémités des microtubules. Cette thèse traite d’abord des concepts mécanistiques sur le cycle catalytique de la kinésine-13. Afin de mieux comprendre le mécanisme moléculaire par lequel les protéines de kinésine-13 induisent la dépolymérisation des microtubules, nous rapportons la structure cristalline d’un monomère de kinésine-13 catalytiquement actif (Kif2A) en complexe avec deux hétérodimères αβ-tubuline courbés dans un réseau tête-à-queue. Nous démontrons également l’importance du « cou » spécifique à la classe de kinésine-13 dans la dépolymérisation catalytique des microtubules. Ensuite, nous avons cherché à fournir la base moléculaire de l’hydrolyse tubuline-guanosine triphosphate (GTP) et son rôle dans la dynamique des microtubules. Dans le modèle que nous présentons ici, l’hydrolyse tubuline-GTP pourrait être déclenchée par les changements conformationnels induits par les protéines kinésine-13 ou par l’agent chimique stabilisant paclitaxel. Nous fournissons également des preuves biochimiques montrant que les changements conformationnels des dimères de tubuline précèdent le renouvellement de la tubuline-GTP, ce qui indique que ce processus est déclenché mécaniquement. Ensuite, nous avons identifié la kinésine de microtubule Kif2C comme une protéine associée à des modèles d’ADN imitant la rupture double brin (DSB) et à d’autres protéines de réparation DSB connues dans les extraits d’œufs de Xenope et les cellules de mammifères. Les cassures double brin d’ADN (DSB) sont un type majeur de lésions d’ADN ayant les effets les plus cytotoxiques. En raison de leurs graves impacts sur la survie cellulaire et la stabilité génomique, les DSB d’ADN sont liés à de nombreuses maladies humaines, y compris le cancer. Nous avons constaté que les activités PARP et ATM étaient toutes deux nécessaires pour le recrutement de Kif2C sur les sites de réparation de l’ADN. Kif2C knockout ou inhibition de son activité de dépolymérisation des microtubules a conduit à l’hypersensibilité des dommages à l’ADN et à une réduction de la réparation du DSB via la jonction terminale non homologue et la recombinaison homologue. Dans l’ensemble, notre modèle suggère que les protéines de kinésine-13 peuvent interagir avec les dimères de tubuline aux extrémités microtubules et modifier leurs conformations, moduler l’étendue des extrêmités tubuline-GTP dans les cellules et déclencher le désassemblage des microtubules. Ces deux modèles pourraient être des clés pour démêler les mécanismes impliqués dans le nouveau rôle de Kif2C dans la réparation de l’ADN DSB sans s’associer à des polymères de microtubules. / Microtubules are long, cylindrical polymers of the proteins α, β tubulin, used in cells to construct the cytoskeleton, the mitotic spindle and axonemes. These hollow polymers are crucial for many cellular functions including intracellular transport and chromosome segregation during cell division. As cells grow, divide, and differentiate, microtubules go through a process, called dynamic instability, which means they constantly switch between growth and shrinkage states. This conserved and fundamental feature of microtubules is tightly regulated by families of microtubule-associated proteins (MAPs). Kinesin-13 proteins are a family of microtubule regulatory factors that catalytically depolymerize microtubule ends. This thesis first discusses mechanistic insights into the catalytic cycle of kinesin-13. In order to better understand the molecular mechanism by which kinesin-13 proteins induce microtubule depolymerization, we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array. We also demonstrate the importance of the kinesin-13 class-specific “neck” in modulating Adenosine triphosphate (ATP) turnover and catalytic depolymerization of microtubules. Then, we aimed to provide the molecular basis for tubulin-Guanosine triphosphate (GTP) hydrolysis and its role in microtubule dynamics. Although it has been known for decades that tubulin-GTP turnover is linked to microtubule dynamics, its precise role in the process and how it is driven are now well understood. In the model we are presenting here, tubulin-GTP hydrolysis could be triggered via the conformational changes induced by kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We also provide biochemical evidence showing that conformational changes of tubulin dimers precedes the tubulin-GTP turnover, which indicates that this process is triggered mechanically. Next, we identified microtubule kinesin Kif2C as a protein associated with double strand break (DSB)-mimicking DNA templates and other known DSB repair proteins in Xenopus egg extracts and mammalian cells. DNA double strand breaks (DSBs) are a major type of DNA lesions with the most cytotoxic effects. Due to their sever impacts on cell survival and genomic stability, DNA DSBs are related to many human diseases including cancer. Here we found that PARP and ATM activities were both required for the recruitment of Kif2C to DNA repair sites. Kif2C knockdown/knockout or inhibition of its microtubule depolymerizing activity led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both non-homologous end-joining (NHEJ) and homologous recombination (HR). Interestingly, genetic depletion of KIF2C, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Altogether, our findings shed light on the mechanisms involved in kinesin-13 catalyzed microtubule depolymerization. Our tubulin-GTP hydrolysis model suggests that kinesin-13 proteins may interact with tubulin dimers at microtubules ends and alter their conformations, modulate the extent of the GTP caps in cells and trigger microtubule disassembly. These two models could be keys to unravel the mechanisms involved in the novel role of Kif2C in DNA DSB repair without associating with microtubule polymers.