Le cycle cellulaire est finement régulé dans le temps et l'espace. Nous avons abordé les aspects dynamiques des interactions que la cycline A2 entretient avec ses partenaires Cdk1, Cdk2 et l'ubiquitine au cours du cycle cellulaire, dans des lignées cellulaires humaines. A cette fin, nous avons eu recours aux approches de FRET (Förster/fluorescence resonance energy transfer) et de FLIM (fluorescence lifetime imaging microscopy). Ceci nous a permis de montrer que les formes ubiquitinylées de la cycline A2 apparaissent principalement sous forme de foyers en prométaphase et se propagent ensuite à l'ensemble de la cellule. En outre, nous avons découvert que l'autophagie participe à la dégradation de cette cycline en mitose. Nous discutons les implications de ces observations quant à un rôle éventuel de la cycline A2 au moment de la formation de l'anneau de constriction, ainsi que de la participation de l'autophagie via cette cycline, dans la réponse aux dommages à l'ADN en mitose. / The cell cycle is finely regulated in time and space. We have studied the dynamical aspect of the interactions between cyclin A2 and its partners Cdk1, Cdk2 and ubiquitin during the cell cycle, in human cell lines. To this aim, we have used FRET (Förster/fluorescence resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) techniques. We have thus shown that ubiquitylated forms of cyclin A2 are detected predominantly in foci in prometaphase, before spreading throughout the cell. Moreover, we have shown that autophagy contributes to cyclin A2 degradation in mitosis. We discuss the implications of these observations regarding a possible role of cyclin A2 when the cleavage furrow forms, and the participation of autophagy in DNA damage response in mitosis.

Foram utilizadas proteínas da famÌlia das GFPs e nanocristais fluorescentes de CdSe/ZnS para caracterização da interação e verificação de transferência de energia por ressonância (FRET) entre estes compostos. Formou-se dois pares doador-receptor onde ora uma proteína figurava como doadora, ora um nanocristal ocupava este papel. Verificou-se que, em ambos os casos, o doador sofre supressão da fluorescência após a formação de complexo com o receptor, complexo este motivado por interação eletrostática e dependente de pH. Foi possível comprovar, através da observação de emissão sensitizada e redução da anisotropia, que entre o par formado por nanocristal com emissão no verde e proteína HcRed1 como receptora, de fato ocorre FRET. As distâncias aparentes entre doador e receptor foram determinadas a partir da eficiência da supressão da fluorescência do doador e da distância de Förster. As distâncias assim obtidas são compatíveis com as dimensões das proteínas e dos nanocristais / Proteins belonging to the GFP family were used to characterize their interaction with fluorescent CdSe/ZnS nanocrystals and to verify the occurrence of resonance energy transfer (FRET) among these elements. Two donor-acceptor pairs were established, one having a protein as donor and the other having a nanocrystal as donor. In both cases the donor suffers quenching of its fluorescence after the formation of a complex with the acceptor. The complex formation is dependent on pH and is due to electrostatic interaction. It was possible to prove the occurrence of FRET between CdSe/ZnS nanocrystals emitting green fluorescence as donors and the protein HcRed1 as acceptor, through the detection of sensitized emission and anisotropy reduction. Apparent donor-acceptor distances were determined from efficiency measurements and Förster distances. The obtained distances agreed with the protein and nanocrystal dimensions

<p>Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.</p>

No description available.

NEDD8, molécule de la famille de l’ubiquitine est essentielle au développement, à la croissance et à la viabilité d’un organisme, de plus c’est une cible prometteuse en thérapeutique. Nous avons découvert que l’inhibiteur spécifique de la NEDDylation, MLN4924 altère la morphologie sans fragmentation et augmente la surface du nucléole de cellules humaines et de noyaux de la lignée germinale de Caenorhabditis elegans. Une approche de protéomique quantitative (SILAC) combiné à l’analyse de la production des ARNr et des ribosomes montrent que MLN4924 change la composition protéique du nucléole sans affecter l’activité transcriptionnelle de l’ARN pol I. Notre analyse montre que MLN4924 active p53 par la voie RPL11/RPL5-Mdm2 caractéristique d’un stress du nucléole. Cette étude identifie le nucléole comme une cible intéressante dans l’utilisation d’inhibiteurs de la NEDDylation et apporte un nouveau mécanisme d’activation de p53 par inhibition de la voie NEDD8.Dans une seconde étude nous avons adapté la méthode de FLIM-FRET (« Fluorescence Lifetime Imaging Microscopy – Förster Resonance Energy Transfer ») à l’étude de la compaction de la chromatine à l’échelle du nanomètre dans un organisme vivant. Le nématode Caenorhabditis elegans s’est révélé être un modèle de choix. Au sein des chromosomes méiotiques, nous avons identifié différentes régions de compaction, de niveau variable par mesure du FRET entre histones fusionnées à des protéines fluorescentes. Par une approche originale d’ARN interférence et injection d’un « extra-chromosome » nous avons défini l’architecture à une nano-échelle de différents états de l’hétérochromatine et montré que cette organisation est contrôlée par les protéines HP1 « Heterochromatin Protein 1 » et SETDB1, une protéine « H3-Lysine 9 methyl transferase ». Nous avons également montré que la compaction de l’hétérochromatine est dépendante des condensines I et II et plus particulièrement la condensine I contrôle l’état faiblement compacté de la chromatine.Nos travaux ont confirmé que C. elegans est un modèle d’intérêt majeur pour l’étude des compartiments nucléaires et parfaitement adapté pour des études pré-clinique. / The ubiquitin-like molecule NEDD8 is conserved and essential for viability, growth and development; its activation pathway is a promising target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human cells and Caenorhabditis elegans germ cells in the absence of nucleolar fragmentation. Through SILAC proteomic analysis and rRNA production, processing and ribosome profiling, we show that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of the NEDDylation pathway and provides a mechanism for p53 activation upon NEDD8 inhibition.Then we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay the nano-scale chromatin compaction in a living organism, the nematode Caenorhabditis elegans. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyl-transferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.We confirm that C. elegans is an interesting model to study nuclear signalling and perfectly adapt to be a platform for pre-clinical studies.

Foram utilizadas proteínas da famÌlia das GFPs e nanocristais fluorescentes de CdSe/ZnS para caracterização da interação e verificação de transferência de energia por ressonância (FRET) entre estes compostos. Formou-se dois pares doador-receptor onde ora uma proteína figurava como doadora, ora um nanocristal ocupava este papel. Verificou-se que, em ambos os casos, o doador sofre supressão da fluorescência após a formação de complexo com o receptor, complexo este motivado por interação eletrostática e dependente de pH. Foi possível comprovar, através da observação de emissão sensitizada e redução da anisotropia, que entre o par formado por nanocristal com emissão no verde e proteína HcRed1 como receptora, de fato ocorre FRET. As distâncias aparentes entre doador e receptor foram determinadas a partir da eficiência da supressão da fluorescência do doador e da distância de Förster. As distâncias assim obtidas são compatíveis com as dimensões das proteínas e dos nanocristais / Proteins belonging to the GFP family were used to characterize their interaction with fluorescent CdSe/ZnS nanocrystals and to verify the occurrence of resonance energy transfer (FRET) among these elements. Two donor-acceptor pairs were established, one having a protein as donor and the other having a nanocrystal as donor. In both cases the donor suffers quenching of its fluorescence after the formation of a complex with the acceptor. The complex formation is dependent on pH and is due to electrostatic interaction. It was possible to prove the occurrence of FRET between CdSe/ZnS nanocrystals emitting green fluorescence as donors and the protein HcRed1 as acceptor, through the detection of sensitized emission and anisotropy reduction. Apparent donor-acceptor distances were determined from efficiency measurements and Förster distances. The obtained distances agreed with the protein and nanocrystal dimensions

Les récepteurs couplés aux protéines G (RCPG) représentent la plus grande famille de protéines membranaires, et ils sont la cible de plus de 25% des médicaments. Ces récepteurs activent diverses voies de signalisation cellulaire via plusieurs familles de protéines G hétéro-trimériques (Gs, Gq, Gi/o et G12/13). Etant donné qu’un RCPG peut activer différentes protéines G, il est important de comprendre comment des ligands favorisent l’activation de certaines protéines G au détriment des autres (ligands biaisés). L’objectif de mon travail a été de développer de nouveaux tests pour l’étude des protéines G qui soient spécifiques d’une famille voire même de certains sous-types de protéines. / G protein-coupled receptors (GPCRs) represent the main family of membrane proteins, and they are the target of more than 25% of drugs in the market. These receptors activate various signaling pathways through different families of heterotrimeric G proteins (Gs, Gq, Gi/o et G12/13). Since a given GPCR can activate several G proteins, it is important to understand how ligands favor the activation of some of these G proteins (biased ligands). The objective of my thesis was to develop assays to study most G protein subtypes.

Mes travaux de thèse portent sur l’interaction de type FRET (tranfert d’énergie résonant de Förster) entre une nanostructure semiconductrice colloïdale individuelle et le graphène. La première partie concerne l’établissement de la théorie du FRET et ce pour plusieurs types de nanostructures. Vient ensuite la partie expérimentale, à commencer par le montage optique ainsi que les méthodes d’analyse, tant pour la spectroscopie que pour la photoluminescence. Par la suite, nous décrivons les résultats obtenus pour divers types de nanocristaux sphériques en interaction directe avec le graphène (incluant des multicouches) : le transfert d’énergie a des effets drastiques sur la photoluminescence mais aussi sur le clignotement des nanocristaux. Puis nous étudions la dépendance du FRET avec la distance ; dans le cas des boîtes quantiques, nous observons une loi en 1/z^4. Par contre, dans le cas de nanoplaquettes, la fonction est plus complexe et dépend de la température. / My PhD subject is the FRET interaction (Förster-like resonant energy transfer) between single colloidal semiconductor nanostructures and graphene. The first part is about the development of the interaction theory with the graphene for several types of nanostructures. Then comes the experimental part, and firstly the optical setup together with the analysis methods, for both spectroscopy and photoluminescence. After that, we describe our results about different types of spherical nanocrystals directly interacting with graphene (which can be multilayer) : the energy transfer has a huge effect on the photoluminescence, as well as the blinking behaviour of the nanocrystals. Then we measure the dependency of the energy transfer as a function the distance ; in the case of quantum dots, we observe a 1/z^4 law. On another hand, in the case of nanoplatelets, the function is more complex and depends on the temperature.

Les récepteurs métabotropes du glutamate (mGluR) sont des récepteurs couplés aux protéines G qui régulent la transmission synaptique. Ce sont des cibles de choix pour le traitement de maladies neurologiques et psychiatriques telles que la maladie de Parkinson et la schizophrénie.J'ai développé une stratégie d'étude de l'assemblage multimérique des protéines membranaires dans des cellules vivantes, à l'aide de techniques de marquage orthogonal et de FRET en temps-résolu. De façon inattendue, j'ai montré que certaines sous-unités de mGluR, en plus de former des récepteurs homodimériques, peuvent former des récepteurs hétérodimériques fonctionnels. D'autre part, j'ai appliqué ces techniques à l'étude du mécanisme d'activation des mGluR et de leur régulation allostérique. J'ai démontré qu'un mouvement relatif des domaines extracellulaires au sein du dimère était responsable de l'action du glutamate.Ce travail a permis de mieux comprendre le fonctionnement des mGluR, et permet la conception de nouveaux tests de criblage. / Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that regulate synaptic transmission. They are relevant therapeutic targets for neurological and psychiatric disorders, such as Parkinson disease and schizophrenia.I developed a strategy to study the multimeric assembly of membrane proteins in living cells, through a combination of orthogonal labeling and time-resolved FRET. Unexpectedly, some subunits of mGluRs, in addition to forming homodimeric receptors, were found capable of forming functional heterodimeric receptors. Then, I applied these techniques to study the activation mechanism of mGluRs and their allosteric regulation. I demonstrated that a conformational change of the dimeric extracellular domain is responsible for the action of glutamate.In addition to increase our understandings of how mGluRs function, this work opens new avenues for the design of drug screening tests.

Le cytosquelette représente un élément crucial dans les processus cellulaires essentiels des cellules lymphoïdes. Les différents filaments du cytosquelette et leurs modes de régulation représentent donc des cibles thérapeutiques majeures pour le développement de nouveaux composés pharmacologiques. Au cours de ce travail de thèse, nous avons mis au point une nouvelle méthode d’analyse par cytométrie en flux (CytoFRET) permettant de visualiser simultanément la dynamique de polymérisation des filaments d’actine, des microtubules et des filaments intermédiaires de vimentine dans la lignée leucémique T Jurkat. Cette méthode a été utilisée pour le criblage d’une mini-chimiothèque composée d’inhibiteurs d’enzymes impliquées dans les modifications post-traductionnelles des protéines. Nous avons ainsi identifié deux composés, le WP1130 et le b-AP15, des inhibiteurs d’enzymes de déubiquitination (DUBs), comme puissants inducteurs de la polymérisation/nucléation de l’actine. Nous avons montré que l’effet de ces inhibiteurs sur les microfilaments d’actine est consécutif à une poly-ubiquitination de la Destrine, une protéine de liaison à l’actine. Nous avons également identifié des inhibiteurs des déacétylases HDAC6 et SIRT2 comme inducteurs de la polymérisation des microtubules et de l’assemblage de la vimentine. L’effet de ces inhibiteurs a été corrélé à une acétylation directe de la tubuline mais pas de la vimentine. Ces résultats ouvrent ainsi de nouvelles perspectives à la fois fondamentale et thérapeutique sur la physiopathologie du cytosquelette des cellules lymphoïdes. / Actin, microtubules, and intermediate filaments compose three major cytoskeletal structures of vertebrate cells that are characterized by highly dynamic balances between assembly and de-assembly, underlying critical cellular processes such as mitosis, architecture and movement. Consequently, cytoskeleton dysfunctions have been implicated in several pathological situations including cell transformation and metastasis. Thus, cytoskeletal networks represent major targets for the development of novel anti-cancer and anti-metastatic therapies. However, drug development is currently limited by the availability of high-throughput screening systems allowing the simultaneous monitoring of actin, microtubules and intermediate filaments dynamics in living cells. In this work, we have developed a novel screening assay of cytoskeleton dynamics based on the simultaneous recording by flow cytometry of FRET signals produced by the variation of actin, tubulin and vimentin filaments dynamics in living cells. Our novel method was employed to screen a mini-library of drugs known for their ability to interfere with post-translationnal modifications of proteins. Interestingly, our approach revealed that compounds interfering with lysine acetylation have a dramatic impact on vimentin filaments assembly and microtubules polymerization. In addition, two inhibitors (WP1130 and b-AP15) of deubiquitinating enzymes showed increase of actin polymerization. This effect was attributed to poly-ubiquitnation of Destrin, an actin binding protein. In conclusion, our FRET multiplex flow cytometry assay represents a novel effective method for the future development of new anti-cancer therapies.