Dissertations / Theses on the topic 'Rag GTPase'
To see the other types of publications on this topic, follow the link: Rag GTPase.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Rag GTPase.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
ESPOSITO, ALESSANDRA. "DIVERSITY IN MTORC1 SUBSTRATE RECRUITMENT ENABLES SPECIFICITY OF METABOLIC RESPONSES TO NUTRITIONAL CUES." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/793428.
Full textAbstract:
The mechanistic target of rapamycin kinase complex 1 (mTORC1) is a key signaling hub that acts as a central regulator of several cellular processes, including cell growth and metabolism. The activation of mTORC1 occurs at the lysosomal surface via a two-step mechanism that requires a) its amino acid-dependent recruitment to the lysosome via the Rag GTPases and b) its growth factor-dependent activation by Rheb.
mTORC1 senses and integrates multiple upstream signals to phosphorylate a broad number of substrates and modulate the crucial balance between cell anabolism and catabolism. However, whether mTORC1 can differentially regulate specific proteins to selectively respond to such a variety of intracellular and environmental cues is poorly understood.
Here we show that Transcription Factor EB (TFEB), a master modulator of lysosomal biogenesis and autophagy, is modulated by mTORC1 via a specific substrate recruitment mechanism that is mediated by Rag GTPases. Differently from the well-characterized mTORC1 substrates S6K and 4E-BP1, which are recruited by mTORC1 via binding to the regulatory subunit Raptor, TFEB interaction with mTORC1 relies on its physical association with active Rag C/D. Owing to this mechanism, TFEB phosphorylation is insensitive to growth factor-mediated activation of Rheb but highly sensitive to amino acid-mediated activation of Rag GTPases. Strikingly, substituting the region of TFEB responsible for its recruitment to mTORC1 with the one of S6K, inverted TFEB phosphorylation behaviour and made it similar to S6K/4E-BP1.
Thus, our findings reveal that diversity in mTORC1 substrate recruitment mechanisms enables mTORC1 to induce selective responses to specific nutritional cues.
2
Belbachir, Nadjet. "Mécanismes physiopathologies du syndrome de Brugada : caractérisation d'un nouveau gène morbide Rad GTPase." Thesis, Nantes, 2017. http://www.theses.fr/2017NANT1015/document.
Full textAbstract:
Le syndrome de Brugada est un trouble du rythme cardiaque héréditaire qui mène à l’apparition de fibrillations ventriculaires et à la mort subite cardiaque. Seulement 30% des cas atteints de ce syndrome sont liés à des mutations génétiques et ce à cause de la complexité du phénotype engendré. Le gène RRAD a été identifié dans une famille qui compte 5 membres atteints du syndrome de Brugada, tous porteurs du variant p.R211H. Ce gène code pour la protéine G monomérique Rad dont le rôle principal est de réguler le courant calcique de type L dans les cellules musculaires squelettiques et cardiaques. Cette étude associe trois modèles d’étude visant à discriminer l’implication de Rad dans le phénotype des patients atteints : Un modèle de surexpression pour étudier le rôle de Rad et l’impact de sa surexpression sur l’activité électrique et la structure des cardiomyocytes, des cardiomyocytes dérivés de cellules IPS reprogrammées des patients porteurs de la mutation pour en déterminer le phénotype cellulaire, et un modèle de souris knock in pour la mutation p.R211H généré dans le but d’intégrer le phénotype cellulaire à l’échelle de l’organe entier. Les résultats obtenus sur les trois modèles, montrent que Rad R211H provoque des troubles au niveau de l’activité électrique du coeur mais aussi au niveau de la structure des cellules différenciées et ces troubles se traduisent par des anomalies à l’ECG chez la souris. Cette étude est la première à démontrer l’implication de Rad GTPase dans le syndrome de Brugada et la seule à démontrer, à ce jour, des perturbations du cytosquelette dans cette pathologie qui est toujours considérée comme une pathologie exclusivement rythmiqueBrugada syndrome (BrS) is a rare inherited cardiac disorder linked to high risk of ventricular arrhythmias and sudden death. In the present day, only 30% of BrS cases have known genetic causes. Most of these mutations have been identified in the SCN5A gene that encodes the cardiac voltage-gated sodium channel NaV1.5. We identified a rare variant in the RRAD gene encoding for the small G protein Rad GTPase, in a familial case of BrS. The aim of this work was to elucidate the mechanisms by which the RRAD p.R211H variant could lead to BrS. First, an overexpressing model was developed using neonatal mouse cardiomyocytes to define the involvement of Rad in the electrical function of cardiomyocytes. Then, cardiac cells were derived from human induced pluripotent stem cells reprogrammed from the carriers of the Rad mutation in order to investigate the phenotype induced at the cellular level. Furthermore, a knock in mouse has been generated to study the impact of this same mutation on the organ level. The three models summarized in a complementary way the phenotype caused by the Rad mutation on the electrical activity at the cellular and the organ levels. The mutation seem to trigger structural defects in the cardiomyocytes that can be involved in the electrical defects related to the disease. The present study is the first report of the potential link between Rad GTPase and BrS. The phenotype reported recapitulates the classical electrophysiological signature of the disease but also associates cytoskeleton disturbances
3
Winge, Per. "The evolution of small GTP binding proteins in cellular organisms. Studies of RAS GTPases in arabidopsis thaliana and the Ral GTPase from Drosophila melanogaster." Doctoral thesis, Norwegian University of Science and Technology, Faculty of Natural Sciences and Technology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-169.
Full textAbstract:
Small GTP binding proteins function as molecular switches which cycles between GTP-bound ON and GDP-bound OFF states, and regulate a wide variety of cellular processes as biological timers. The first characterized member of the small GTPase family, the mutated oncogene p21 src, later known as Harvey-Ras, was identified in the early 1980s (Shih, T. Y. et al. 1980). In the following years small Ras-lik GTPases were found in several organisms and it was soon discovered that they took part in processes, such as signal transduction, gene expression, cytoskeleton reorganisation, microtubule organisation, and vesicular and nuclear transport. The first Rho (Ras homology) gene was cloned in 1985 from the sea slug Aplysia (Madaule, P. et al. 1985) and because of their homology to Ras it was first suspected that they could act as oncogenes. Later studies have shown that even though they participate in processes such as cell migration and motility they are not mutated in cancers.
The first indications that Rho was a signaling protein regulating the actin cytoskeleton, came from experiments where activated forms of human RhoA was microinjected into 3T3 cells (Paterson, H. F. et al. 1990). Another Rho-like GTPase Rac1 (named after Ras-related C3 botulinum toxin substrate) was later shown to regulate actin cytoskeletal dynamics as well, suggesting that Rho-family members cooperate in controlling these processes (Ridley, A. J. et al. 1992). The Rac GTPase was also implicated in regulating the phagocytic NADPH oxidase, which produce superoxide for killing phagocytized microorganisms (Abo, A. et al. 1991). Thus, it soon became clear that Rac/Rho and the related GTPase Cdc42 (cell division cycle 42) had central functions in many important cellular processes.
There are at least three types of regulators for Rho-like proteins. The GDP/GTP exchange factors (GEFs) which stimulates conversion from the GDPbound form to the GTP-bound form. GDP dissociation inhibitors (GDIs) decrease the nucleotide dissociation from the GTPase and retrieve them from membranes to the cytosol. GTPase activating proteins (GAPs) stimulates the intrinsic GTPase activity and GTP hydrolysis. In addition there are probably regulators that dissociate GDI from the GTPase leaving it open for activation by the RhoGEFs.
Ras and Rho-family proteins participate in a coordinated regulation of cellular processes such as cell motility, cell growth and division. The Ral GTPase is closely related to Ras and recent studies have shown that this GTPase is involved in crosstalk between both Ras and Rho proteins (Feig, L. A. et al. 1996; Oshiro, T. et al. 2002). Ral proteins are not found in plants and they appear to be restricted to animalia and probably yeast. During a screen for small GTPases in Drosophila melanogaster I discovered in 1993 several new members of the Ras-family, such as Drosophila Ral (DRal), Ric1 and Rap2. The functions of Ral GTPases in Drosophila have until recently been poorly known, but in paper 2 we present some of the new findings.
Rho-like GTPases have been identified in several eukaryotic organisms such as, yeast (Bender, A. et al. 1989), Dictyostelium discoideum (Bush, J. et al. 1993), plants (Yang, Z. et al. 1993), Entamoeba histolytica (Lohia, A. et al. 1993) and Trypanosoma cruzi (Nepomuceno-Silva, J. L. et al. 2001). In our first publication, (Winge, P. et al. 1997), we describe the cloning of cDNAs from RAC-like GTPases in Arabidopsis thaliana and show mRNA expressions pattern for five of the genes. The five genes analyzed were expressed in most plant tissues with the exception of AtRAC2 (named Arac2 in the paper), which has an expression restricted to vascular tissues. We also discuss the evolution and development of RAC genes in plants. The third publication, (Winge, P. et al. 2000), describe the genetic structure and the genomic sequence of 11 RAC genes from Arabidopsis thaliana. As most genomic sequences of the AtRACs we analyzed came from the Landsberg erecta ecotype and the Arabidopsis thaliana genome was sequenced from the Columbia ecotype, it was possible to compare the sequences and identify new polymorphisms. The genomic location of the AtRAC genes plus the revelation of large genomic duplications provided additional information regarding the evolution of the gene family in plants. A summary and discussion of these new findings are presented together with a general study of small Ras-like GTPases and their evolution in cellular organisms. This study suggests that the small GTPases in eukaryots evolved from two bacterial ancestors, a Rab-like and a MglA/Arp-like (Arf-like) protein. The MglA proteins (after the mgl locus in Myxococcus xanthus) are required for gliding motility, which is a type of movement that take place without help of flagella.
The second publication describes experiments done with the Drosophila melanogaster DRal gene and its effects on cell shape and development. Ectopic expression of dominant negative forms of DRal reveals developmental defects in eye facets and hairs, while constitutive activated forms affects dorsal closure, leaving embryos with an open dorsal phenotype. Results presented in this publication suggest that DRal act through the Jun N-terminal kinase (JNK) pathway to regulate dorsal closure, but recent findings may point to additional explanations as well. The results also indicate a close association between processes regulated by Rac/Rho and Ral proteins in Drosophila.
4
Ghiaur, Gabriel. "The role of Rho GTPases in hematopoietic stem cell biology RhoA GTPase regulates adult HSC engraftment and Rac1 GTPases is important for embryonic HSC /." Cincinnati, Ohio : University of Cincinnati, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204374567.
Full text
5
Maximano, Filipe Manuel Correia. "Armus : A novel link between Rac and Rab small GTPases." Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526397.
Full text
6
Yarwood, Sam. "Calcium signalling and the small GTPase Ras." Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492619.
Full textAbstract:
Calcium (Ca ²?) is a highly versatile signal that regulates a host of intracellular events across the biological spectrum. Carefully regulated changes in intracellular Ca²? concentration, over a broad temporal and spatial range, carry complex signals to a plethora of proteins who decode and transduce the information and regulate a wide variety of physiological processes.
7
Kirsten, Marie Lis. "Biophysical studies of Rab GTPase membrane binding." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6964.
Full textAbstract:
Rab proteins are the largest subfamily of the Ras superfamily of small GTPases, with more than 60 known members, that are involved in a multitude of different processes regulating membrane traffic. Rab proteins cycle between the cytosol and association with membranes, whereby each Rab exhibits a characteristic and specific subcellular localisation. It remains obscure how Rab proteins, in spite of high sequence and structure identity, distinguish between different membranes in the cell with such specificity. Membrane biophysical properties, such as stored curvature elastic stress and bending rigidity, are increasingly found to be determinants for protein recruitment and activity, and other Ras related proteins have recently been shown to exhibit sensitivity towards lipid species and elastic membrane properties. In this study Rab membrane binding is for the first time correlated to membrane bending rigidity, suggesting that biophysical properties of lipid membranes may play a role in the regulation of Rab targeting. Furthermore, all Rab proteins tested were observed to bind membranes in the absence of other protein factors, questioning the function of protein targeting factors for the Rab membrane recruitment process. Another aspect of Rab membrane interaction is Rab extraction from membranes by GDI. A large scale in vitro screening of 17 Rab proteins revealed a broad range of extractability from membranes with GDI. No correlation was found between extractability and the C-terminal prenylation motif, and no difference in extractability was observed in direct comparison of the extraction potential with GDIα and β. However, Rab proteins that exhibited low extractability from membranes are involved in secretory processes, suggesting a functional correlation to extractability. Furthermore, Rab40c as the first mammalian Rab protein to date was shown to be palmitoylated.
8
Wakade, Rohan Sanjay. "Rôle de GTPase de type Rab, Ypt6, chez le pathogène fongique opportuniste de l’homme, Candida albicans." Thesis, Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4064.
Full textAbstract:
Candida albicans est un organisme commensal présent dans le microbiote, qui peut cependant provoquer des infections superficielles mais aussi systémiques, engageant alors le pronostic vital chez les patients immunodéprimés. La transition entre forme bourgeonnante et forme filamenteuse hyphale hautement polarisée, ce qui nécessite une réorganisation du cytosquelette et un trafic membranaire soutenu, est associée à la virulence. Chez les eucaryotes, les GTPases de la famille Rab (Ras related protein in the brain) et leurs régulateurs jouent un rôle central dans le trafic membranaire. L'objectif de ce travail est de comprendre le rôle de ces protéines, en particulier de Ypt6, l'homologue de Rab6 humain, dans la transition morphologique et la virulence de C. albicans. Dans ce but, j'ai construit des mutants « perte de fonction » et déterminé que YPT6 n'est pas essentiel à la viabilité, mais est critique pour l'intégrité de la paroi cellulaire et la croissance hyphale invasive ; les hyphes du mutant ypt6 sont plus courtes que celles de la souche sauvage. En outre, YPT6 est critique pour la virulence dans deux modèles murins de candidose. Lors de la croissance hyphale, Ypt6 est co-localisé avec Arl1, une GTPase de la famille Arf (ADP Ribosylation Factor), également nécessaire pour la croissance hyphale et la virulence de C. albicans. De plus, la surexpression de YPT6 compense spécifiquement le défaut de croissance hyphale du mutant de délétion arl1, mais pas l'inverse. La délétion de YPT6 résulte également en une augmentation du nombre de citernes Golgiennes, suggérant que l'intégrité du Golgi est altérée dans ce mutant. Utilisant de l'imagerie sur cellules vivantes, j'ai montré que la distribution d’Abp1 (Actin binding protein 1), qui est un rapporteur des sites d’endocytose, est aussi altérée dans le mutant ypt6, en ceci qu’elle n’est plus restreinte à l’apex de l’hyphe, comme observé dans les cellules sauvages. Ces données suggèrent que le défaut de maintien de la croissance hyphale du mutant ypt6 est au moins en partie associé à une altération de la distribution des sites d’endocytose. En résumé, j’ai identifié le rôle de Ypt6 dans la croissance hyphale invasive et la virulence du pathogène fongique opportuniste de l’homme C. albicans, et mis en évidence une interaction entre deux GTPases, Ypt6 et Arl1, lors du processus de croissance hyphaleCandida albicans is a harmless constituent of the human microbiota that causes superficial infections as well as life threatening infections in immune compromised individuals. The transition from a budding form to the highly polarized hyphal form is associated with virulence and requires cytoskeleton reorganization and sustained membrane trafficking. In a range of eukaryotes, Ras related protein in the brain (Rab) G proteins and their regulators have been shown to play a central role in membrane traffic. The objective of this work is to understand the role of Rab proteins, in particular Ypt6, the homolog of Human Rab6, in the morphological transition and virulence of C. albicans. To this aim, I generated loss of function mutants and found that YPT6 is not essential for viability, yet was critical for cell wall integrity and invasive hyphal growth, with ypt6 hyphal filaments shorter compared to that of the wild type (WT). Furthermore, YPT6 was important for virulence in two murine candidiasis models. I determined that Ypt6 was localized at the late Golgi compartment during hyphal growth, where it co-localized with Arl1, a small GTPase of the Arf (ADP Ribosylation Factor) family, also required for hyphal growth and virulence. Interestingly, overexpression of YPT6 specifically rescued the hyphal growth defect of the arl1 mutant, but not the converse. Further characterization of the ypt6 deletion mutant showed that the number of Golgi cisternae is increased in this mutant compared to that of WT strain, suggesting an alteration of Golgi integrity. In addition, using live cell imaging I showed that the distribution of Actin binding protein 1 (Abp1), which is a reporter for actin patches, was altered in the ypt6 mutant, in that it was no longer restricted to the tip of the filament, as is observed in WT cells. These data suggest that the defect in hyphal growth maintenance of the ypt6 deletion mutant is at least partly associated with an alteration of the distribution of endocytic sites. Thus, I identified a critical role of Ypt6 during invasive hyphal growth and virulence in the human fungal opportunistic pathogen C. albicans and revealed an interaction between Ypt6 and Arl1 in the hyphal growth process
9
Fan, Wing-Tze. "Characterization of Ras-GRF2, a bifunctional guanine nucleotide exchange factor for the Ras and Rac GTPases." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ63720.pdf.
Full text
10
Jilkina, Olga. "The function of Ral GTPase in human platelets." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ41614.pdf.
Full text
11
Lumb, Jennifer. "Exploration of Rab GTPase function in Trypanosoma brucei." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608669.
Full text
12
Aresta, Sandra. "Etude de la protéine Gem, un membre de la superfamille Ras." Paris 11, 2001. http://www.theses.fr/2001PA112281.
Full textAbstract:
Gem est une nouvelle protéine de la superfamille Ras (famille RGK) dont l'expression est induite dans différents types cellulaires après stimulation par des agents mitogènes. Gem possède des extensions N-terminale et C-terminale par rapport à Ras de fonctions inconnues, ainsi que des substitutions d'acides aminés dans des positions clés pour la liaison et hydrolyse du GTP. Afin de rechercher les partenaires cellulaires de Gem, est plus particulièrement ceux de cette région N-terminale, nous avons mis au point un système double hybride modifié où la protéine appât est fusionnée en position N-terminale par rapport au domaine de liaison à l'ADN de la protéine bactérienne LexA, c'est-à-dire, de polarité inversée à celle des systèmes double-hybride classiques. Nous l'avons validé en montrant qu'il conservait la spécificité d'interaction entre les GTPases Ras et Ral et ses régulateurs et effecteurs, et possédait même dans certains cas une sensibilité de détection accrue. Nous avons construit une banque d'ADNc à partir de cellules de la lignée de lymphome T humain Jurkat, que nous avons criblée en utilisant comme appât : (i) les 82 acides aminés N-terminaux de Gem fusionnés à l'extrémité N-terminale du domaine de liaison à l'ADN (DBD) de LexA ; (ii) la protéine Gem entière fusionnée à l'extrémité C-terminale du DBD de GAL4. Parmi les 20 clones positifs obtenus contenant des ADNc distincts, nous avons décidé d'en étudier deux. Ces ADNc codent pour une protéine homologue à la protéine 4. 1N, et une nouvelle protéine avec un domaine RhoGAP. Nous avons mis en évidence que la nouvelle protéine à domaine RhoGAP est bien capable d'augmenter l'activité GTPase intrinsèque de GTPases de la sous-famille Rho/Rac/Cdc42, aussi bien in vitro que in vivo, et nous avons démontré par coimmunoprécipitation que Gem est capable d'interagir avec cette protéine dans des cellules eucaryotes. Ces données suggèrent que Gem pourrait jouer un rôle dans la régulation de la fonction des protéines RhoGem is a recently identified protein belonging to the branch RGK of the Ras superfamily of GTPases. Gem is induced in several cell types upon mitogen stimulation, and it presents N- and C-terminal extensions of unknown function when compared to Ras as well as several amino acid substitutions in key positions for GTP binding and hydrolysis. With the aim of identifying Gem partners, and in particular those interacting with its N-terminal extension, we have developed a new vector for two-hybrid studies where the bait is fused through its C-terminus with the N-terminus of the DNA-binding domain (DBD) of LexA, therefore, possessing an inverted polarity as compared with a classical two-hybrid vector. We have validated this system by showing that it allows the same specific interactions between Ras and Ra1 GTPases with their effectors and regulators as a classical two-hybrid vector, and that it even shows an increased sensibility. We have built a two-hybrid cDNA library from Jurkat cells that we have screened for Gem partners with two different baits: (i) the first 82 amino acids of Gem fused to the N-terminus of LexA DBD; (ii) full-length Gem fused to the C-terminus of GAL4 DBD. Amongst the 20 different clones obtained, two have been studied further. Their cDNAs coded for an isoform of the 4. 1N protein, and for a novel protein containing a RhoGAP domain. We have shown that the new RhoGAP protein is capable of increasing the intrinsic GTPase activity of GTPases belonging to the Rho/Rac/Cdc42 branch, in vitro as well as in vivo, and we have demonstrated by co-immunoprecipitation that Gem is able to interact with this protein in eukaryotic cells. These results suggest that Gem could be implicated in the regulation of Rho GTPases function
13
Bettoun, Audrey. "The NDR1 Kinase, a New Player in Oncogenic Signalling of Ral GTPases, Functions as a Linchpin Between Cancer Cell Survival and Death." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA11T047.
Full textAbstract:
Des mutations du gène Ras jouent un rôle essentiel dans le développement tumoral. Les GTPases Ral , RalA et RalB, sont des effecteurs proximaux de l’oncogène Ras. RalA permet la croissance en absence de substrat et RalB est nécessaire à l'autophagie et à la résistance à l'apoptose des cellules cancéreuses. Cette thèse a pour objectif de clarifier les mécanismes moléculaires de la signalisation Ral impliqués dans l’oncogenèse dépendante des protéines Ras.Des criblages par double hydride ont été effectués par notre équipe et un interactome de Ral a été établi. Ce criblage a montré une interaction entre des protéines de la signalisation Ral et la protéine NDR1, une kinase pro-apoptotique appartenant à la voie " suppresseur de tumeur" Hippo. Le Projet 1 montre la régulation de NDR1 par la voie RalA-Exocyste- MAP4K4 en réponse au stress osmotique, oxydatif ou au traitement par le TNF-α. Dans cette voie, la kinase MAP4K4, un effecteur de RalA, via le complexe exocyste active directement NDR1. En outre, nous avons montré que la voie RalA-MAP4K4-NDR1 était nécessaire à l'apoptose déclenchée par le TNF-α ou par la surexpression de RASSF1A, suppresseur de tumeur appartenant à la voie Hippo. Nous avons donc montré que RalA a un rôle pro-apoptotique inattendue qui agit via la kinase NDR1, en plus de son rôle connu de proto-oncogène en aval de Ras.Le projet 2 montre que la protéine kinase NDR1 est un régulateur de l'autophagie. Des criblages par double hydride ont été effectués par notre équipe avec NDR1 comme appât et ont permis de montrer une interaction entre Beclin 1, une protéine majeure de l’autophagie, et NDR1. Nous avons montré que NDR1 était nécessaire à l'autophagie et à la formation des autophagosomes chez l'humain et la Drosophile. De plus, NDR1 est nécessaire à la formation du complexe Exo84 de l'exocyste, Beclin1 et RalB nécessaire à l'initiation de l'autophagie. Nous montrons également que RalB régule l'état d'activation de NDR 1 après induction de l'autophagie. En effet, en absence de RalB, nous avons observé une hyper - activation de NDR1 menant les cellules vers l'apoptose. Ainsi nous avons montré que NDR1 joue le rôle d'interrupteur favorisant l'autophagie ou favorisant l'apoptose suivant son état d'activation.Le projet 3 étudie l'implication de la voie RalGTPases-NDR1 dans l'oncogenèse dépendante de Ras et dissèque par quels mécanismes NDR1 y contribueConstitutive Ras signalling is one of the most frequent oncogenic event in human cancers. Thus, it is imperative to identify new therapeutic options targeting downstream effectors of Ras signalling. Ras-like GTPases RalA and RalB are proximal effectors of oncogenic Ras. RalA was reported to support anchorage independent proliferation and RalB regulates autophagy and inhibits apoptosis of cancer cells. Ral proteins execute these functions via several direct effectors as the exocyst, an octameric complex originally identified as regulator of vesicles trafficking. The global goal of this PhD was to better decipher the molecular mechanisms underlying the functions of Ral GTPases in oncogenesis.To extend the Ral interactome, i.e. the protein-protein interaction network centered on Ral, we performed yeast-two hybrid screenings which led to the identification of the NDR1 kinase, belonging to the tumor suppressor Hippo pathway. NDR1 functions in oncogenesis were investigated in the context of three projects.In Project 1, we showed that NDR1-dependent apoptosis is regulated by a RalA/Exocyst/MAP4K4/NDR1 cascade. We reported that under osmotic or oxidative stresses or TNF-α treatment, the Ste20-like MAP4K4 kinase, an effector of RalA via the exocyst complex, directly activates NDR1. Moreover, we found that TNF-α treatment or overexpression of the tumor suppressor RASSF1A, which belongs to the Hippo pathway, leads to apoptosis through this RalA/Exocyst/MAP4K4/NDR1 pathway. This novel and unexpected pro-apoptotic role of RalA suggests that the RalA GTPase can positively signal in tumor suppressor pathways via the kinase NDR1, in addition to its proto-oncogenic role downstream of Ras. In Project 2, we described the NDR1 protein kinase as a conserved regulator of autophagy. Using NDR1 as bait in yeast two hybrid screens, we fished Beclin1, a key regulator of autophagy, and we validated the existence of a direct biochemical NDR1-Beclin1 interaction. We showed that NDR1promotes autophagosome formation in human cells and Drosophila larvae. Furthermore, we observed that NDR1 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Very interestingly, under prolonged autophagy, RalB depletion triggers hyperactivation of NDR1 resulting in NDR1-dependent apoptosis. Thus, it appears that the NDR1 kinase could act as a switch between autophagy (=survival) or apoptosis (=death), under the control of RalB. In Project 3, we addressed the role of the newly identified RalGTPases-NDR1axis in Ras - induced oncogenesis and tumorigenesis
14
Mirey, Gladys. "Approche moleculaire et genetique de la cascade des gtpases ras-ral le cas de dral-gef, homologue chez drosophila melanogaster des facteurs d'echange des petites gtpases ral." Paris 11, 2000. http://www.theses.fr/2000PA112116.
Full textAbstract:
La fonction biologique de la gtpase ral n'est pas connue. L'hypothese est que, apres activation d'un recepteur tyrosine kinase, ras est activite et interagit avec le facteur d'echange de ral, qui active ral. Dans un premier temps, les residus specifiant l'interaction de ral, versus ras, avec ses effecteurs, ont ete determines. Une deuxieme approche, associant les resultats d'un crible double-hybride et l'approche genetique chez drosophila melanogaster, a permis d'aborder in vivo la fonction de ral. Le crible double-hybride avec l'appat d-ral a permis de cloner plusieurs adnc codant pour des partenaires de cette gtpase. Les adnc codant pour le facteur d'echange d-rgl et l'effecteur d-rlip ont ainsi ete isoles. L'expression de dral, drgl et drlip a ete etudiee par hybridation in situ ainsi que par immunofluorescence sur embryons et disques imaginaux de drosophile. Le gene rgl a ete localise au niveau chromosomique et sa structure genomique a ete determinee. Deux souches independantes de drosophile, contenant un element p dans le gene rgl, ont ete caracterisees. Un crible d'excision d'element p a permis de generer un mutant pour lequel les exons 1 et 2 de rgl1 sont deletes. Le gene ral est localise sur le chromosome x dans une region depourvue d'insertion d'element p. Une approche par surexpression a donc ete utilisee. Differentes lignees transgeniques permettant l'expression de formes mutees de ral ou de differents alleles de rgl ont ete etablies. L'expression d'un ral inactif ou d'un rgl constitutivement actif de facon ubiquitaire est letale alors que l'expression de ces alleles dans le thorax de la drosophile induit une desorganisation generale des soies, organes sensoriels de la drosophile. Ces mutants permettront d'etablir les relations d'epistasie entre ras, ral et rgl et de determiner la contribution de ral a la voie ras, ainsi que les relations genetiques entre cette voie et d'autres voies de signalisation.
15
Ni, Wenjun. "Involvement of Rac GTPase in p53-deficiency mediated lymphomagenesis." Cincinnati, Ohio : University of Cincinnati, 2006. http://rave.ohiolink.edu/etdc//view?acc_num=ucin1155831060.
Full text
16
Wersäll, Andreas. "Platelet Ral GTPases : an investigative study." Thesis, University of Bristol, 2018. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.761241.
Full text
17
Kulakowski, Guillaume. "Membrane binding properties of RAB GTPases." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066358/document.
Full textAbstract:
Les RAB GTPases sont des régulateurs majeurs du trafic vésiculaire et sont localisées sur des compartiments spécifiques. L'identification des processus moléculaires régulant la localisation des RAB est donc cruciale afin de comprendre les mécanismes de transport intracellulaire. Nous sommes parvenus, pour la première fois, à incorporer des protéines RAB purifiées et prénylées dans des membranes artificielles. Nous avons tout d'abord observé que RAB6 est capable de promouvoir une agrégation de vésicules, phénomène qui n'est pas observé avec RAB1 et RAB5. Nous suggérons un modèle dans lequel RAB6 interagit en trans avec lui-même et par conséquent induit un accolement de membranes. La partie principale de cette étude consistait à identifier les propriétés physicochimiques des membranes requises pour le recrutement des protéines RAB. Nous avons observé que RAB1, RAB5 et RAB6 se lient préférentiellement à des membranes désordonnées et courbées, phénomène qui s'explique par l'insertion du groupement prenyl hydrophobe au niveau de défauts d'agencement de lipides. En revanche, le recrutement de RAB35 requiert la présence de lipides chargés négativement et peut être modulé, dans une moindre mesure, par les défauts d'agencement lipidique. Bien que RAB4 et RAB11 soient recrutées sur des fractions de Golgi purifiées, les charges membranaires et les défauts d'agencement lipidique ne sont pas suffisants pour permettre leur recrutement sur des vésicules synthétiques. Cela suggère que le recrutement de RAB4 et RAB11 nécessite des propriétés membranaires plus complexes. Nos travaux démontrent que les propriétés membranaires sont cruciales pour la localisation spécifique des protéines RABRAB GTPases are major regulators of vesicular trafficking and localize to specific compartments. Deciphering the molecular mechanisms governing RAB localization is thus critical to understand intracellular transport processes. We have managed, for the first time, to incorporate purified and prenylated RABs into artificial membranes. By doing so, we observed that RAB6, but not RAB1 or RAB5, is able to promote by itself vesicle tethering. We believe that RAB6 is able to interact in trans with itself and to consequently drive homotypic membrane tethering. In the main part of this study, we investigated the physicochemical membrane requirements necessary for RAB recruitment. RAB1, RAB5 and RAB6 were all found to only localize to disordered membrane domains and to preferentially bind to curved membranes. We demonstrated that this specific recruitment of RAB1, RAB5 and RAB6 is primarily dependent on the hydrophobic insertion of their prenyl group into lipid packing defects. In contrast, RAB35 recruitment was primarily dependent on the presence of negatively charged lipids and was found to be modulated, to a lesser extent, by lipid packing defects. Although RAB4 and RAB11 were effectively recruited to purified Golgi fractions, in an effector-independent manner, membrane charges and lipid packing defects were not sufficient to promote their recruitment to synthetic vesicles; suggesting that RAB4 and RAB11 require more demanding membrane physicochemical properties. Our work demonstrates that the properties of membranes are critical for the regulation of RAB specific membrane targeting
18
Blessenohl, Marco. "FT-IR-spektroskopische Untersuchungen des Mechanismus der H-ras-p21-katalysierten GTPase." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=967920256.
Full text
19
CHEN, XIAOJING. "Control of Secondary Granule Release in Neutrophils by Ral GTPase." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/96.
Full textAbstract:
Neutrophil (PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocyto-sis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, I found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules, but not primary or tertiary granules, and caused dy-sregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, while chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Over-expression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, I found that Ral, which was associatedwith the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral transloca-tion intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattrac-tant stimulation.
20
Gómez, de la Cuesta Ramón. "Mechanism of regulation of the Ras-GTPase activating protein CAPRI." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611173.
Full text
21
Nachury, Maxence. "La GTPase Ran : un marqueur du génome dans l'espace cellulaire." Paris 11, 2001. http://www.theses.fr/2001PA112257.
Full textAbstract:
La GTPase Ran est impliquée dans plusieurs processus cellulaires essentiels pour l'expression et la propagation du génome eucaryote. La protéine RCCl responsable de la conversion RanGDP->RanGTP est constitutivement liée aux chromosomes alors que la protéine RanGAP qui augmente l'activité GTPase de Ran est cytoplasmique. En conséquence, RanGTP n'est présent que dans la proximité de l'ADN génomique, i. E. Noyau en interphase et zone perichromatinienne en mitose. Le transport de protéines (cargos) entre le cytoplasme et le noyau est un processus actif qui requiert la présence de Ran et de récepteurs de transport. J'ai montré que la translocation de complexes cargo-rocépteur au travers du complexe du pore nucléaire (NPC) ne nécessite pas d*hydrolyse de GTP par Ran. De plus, la présence d'une forte concentration de RanGTP dans le cytoplasme suffit à inverser la direction du transport à travers le NPC. Ces expériences suggèrent que le NPC a le rôle d'un canal qui autorise la diffusion bidirectionnelle de complexes cargo-récepteur. .The Ran GTPase is involved in a number of processes required for the xpression and, maintenance of the eukaryotic genome. Two factors ensure that GTP-bound Ran is only found in the vicinity of the genomic DNA. First, RCC1, the guanine nucleotide exchange factor for Ran, binds tightly and constitutively to chromatin. Second, RanGAP, the GTPase-activating protein for Ran. Is found only in the cytosol. The aim of my thesis research was to understand how this anisotropic distribution of RanGTP orchestrates nucleocytoplasmic transport during interphase and spindle assembly during, mitosis. The transport of proteins into and out of the nucleus is an active process that depends on Ran and specific transport receptors. I found that the translocation of receptor-cargo complexes across the nuclear pore complex (NPC) does not require GTP hydrolysis by Ran. Furthermore, the presence of high concentrations of RanGTP in the cytoplasm can invert the direction of transport through the NPC. .
22
Haas, Alexander. "Regulation of endocytosis and secretion by Rab GTPase activating proteins." Diss., kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/8660/.
Full text
23
Hazelett, C. Clayton. "Ral GTPases regulate biogenesis of cell polarity." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2889.
Full textAbstract:
Cell polarity is the asymmetric distribution of organelles that almost all cells use to separate individual processes and perform complex functions. Although the manner in which cells are polarized is very diverse, the processes necessary to assume polarized phenotypes are similar in many cell types. Epithelial cell polarization is of particular importance, as these cells serve form linings of organs and act as barriers distinguishing different compartments. Furthermore, loss of epithelial polarization occurs in some disease states and may result in cell invasion through underlying matrix. During initial polarization, vesicle trafficking is indispensible for assembly of structures, including apical junctional complex formation. Trafficking of new membrane and associated proteins to leading edges is also necessary for cell migration. RalA and RalB are members of the Ras superfamily of GTPases and have been implicated in several processes, including vesicle trafficking. Only 5 Ral effectors have been identified, two of which are members of the Exocyst complex, a hetero-octameric complex also involved with vesicle trafficking. I hypothesized that Ral GTPases were necessary for several aspects of cell polarization, and that they engage the Exocyst complex to mediate these processes. Initial investigation of tight junction assembly found that both RalA and RalB antagonistically affect paracellular permeability. Knockdown of RalA and RalB resulted in decreased and increased incorporation of components into assembling tight junctions, respectively. Furthermore, both RalA and RalB engaged the Exocyst in order to mediate tight junction assembly. I next examined the role of RalA-Sec5 and RalA-Exo84 interactions during tumor cell migration and invasion. Both interactions were necessary for invasion and single cell migration, although disruption of each interaction affected different aspects of migration. Furthermore, significant differences in cytoskeleton organization occurred in response to disruption of RalA-Sec5 and RalA-Exo84 interactions. Finally, I investigated the effects of RalA and RalB knockdown on growth of primary cilia and cyst formation. RalA decreased primary cilia growth and reduced average cilia length, while RalB increased cilia length. Knockdown of RalA and RalB also affected lumen formation during cystogenesis, as RalA knockdown prevented lumen formation and RalB knockdown caused formation of multiple lumens. Taken together, data presented here show that Ral engages the Exocyst to mediate distinct processes during tight junction assembly and cell migration, and implicates Ral GTPases in several different aspects of cell polarity.
24
Taylor, Adam. "The role of Rab GTPases in osteoclasts." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=59017.
Full text
25
Hanker, Ariella Binah Der Channing J. "Ras family GTPases involved in breast cancer." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2867.
Full textAbstract:
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.Title from electronic title page (viewed Jun. 4, 2010). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular Biology." Discipline: Genetics and Molecular Biology; Department/School: Medicine.
26
Lau, Sin-nga, and 劉善雅. "The role of RAB(rat sarcoma-related proteins in brain) Gtpases in regulating testicular junction dynamics." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31245535.
Full text
27
SADOU, AMEL. "CROSS-TALK BETWEEN RAL AND RAC PATHWAYS IN THE CONTROL OF CELL MIGRATION." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/214614.
Full textAbstract:
SUMMARY (English)
Very little is known about the coordination and the integration among the different regulators of the motility process. This work deals with two migration-regulatory pathways: the Rac1/WRC (Wave Regulatory Complex) pathway that drives the formation of the actin polymerization network at the front of motile cells; and RalB/exocyst pathway for which the molecular mechanisms underlying its implication in cell motility were still largely unknown at the beginning of this thesis. Rac1 and RalB are small GTPases of the Rho and Ras family, respectively. WRC and exocyst complexes are their direct effectors.
In searching for connections between the exocyst and migration regulators, we found that two subunits of the exocyst, Exo70 and Sec6, interact directly in vitro with two subunits of the WRC, Abi and Cyfip, respectively. Moreover, we found that exocyst subunits can interact in vitro with the whole fully-assembled WRC complex. We also showed that these two complexes associate in vivo. Functionally, the exocyst was required for WRC complex positioning at the front of migrating cells.
On the other hand, we also found that two other subunits of the exocyst, Sec8 and Exo84, interact with SH3BP1 (a RhoGAP protein) by two-hybrid assay and by co-immunoprecipitation. SH3BP1 localizes at the leading edge and this localization is dependent on the exocyst. Interestingly, in vivo, the RalB/exocyst/SH3BP1 pathway specifically targets Rac1, and not Cdc42. By a combination of approaches we concluded that SH3BP1 is required to inactivate Rac1 at the front.
In our model we propose that RalB/exocyst regulates cell migration by driving to the leading edge two key signaling elements of the Rac1 pathway: its effector WRC, that stimulates actin filament nucleation, and its negative regulator SH3BP1, a GAP promoting Rac1 inactivation and GDP/GTP cycling. In conclusion, this work provides novel molecular and functional links between polarized exocytosis and actin dynamics during cell motility.
28
Naim, Adnan. "The Role of G3BPs in the Stress Response Pathway." Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367499.
Full textAbstract:
The ras-GTPase SH3-domain Binding Proteins (G3BP) are a family of RNA-binding proteins that have been implicated in multiple cellular activities ranging from signal transduction to regulation of messenger RNA (mRNA). G3BPs were named after their interaction with the SH3 domain of Ras-GTPase-activating protein; however recent research did not find this interaction. All three members of the G3BPs family, G3BP1, G3BP2a and G3BP2b, share structural similarities with each other by having four distinct regions (1) the Nuclear Transporting Factor 2, (NTF2) domain at the N-terminal, (2) the acidic and proline-rich domain in the centre, (3) the RNA recognition motif (RRM) and (4) the arginine glycine (RGG)-rich region rich at the C-terminal. The presence of the NTF2 domain in its structure suggests G3BP might play a role in nucleocytoplasmic transportation, which was observed after serum stimulation where G3BP1 was translocated to the nucleus from the cytoplasm. The RNA recognition motif (RRM) region plays a vital role in its interaction with the target RNA. The RGG-rich box is a region rich in arginine and glycine residues, which plays a role assisting RRM in interactions with protein or RNA.
G3BP1 is found to be overexpressed in many cancers, including breast cancer, and head and neck tumours, as well as cell lines derived from human lung, prostrate, colon, thyroid and breast cancer. G3BPs have also been implicated in translational control within differentiating neurons, suggesting that G3BP may play several roles in controlling the translational fate of its cargo and that its role may be cell-specific. G3BP1 has also been found in β-integrin- induced adhesion complexes. This information highlights G3BPs as a dynamic protein that is involved in several biological functions.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
Full Text
29
Smith, Harvey W. "Signalling from uPAR to the Activation of the Small GTPase Rac." Thesis, Institute of Cancer Research (University Of London), 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499157.
Full text
30
Linford, Andrea. "The identification of Rab GTPase control systems involved in cell migration." Thesis, University of Liverpool, 2012. http://livrepository.liverpool.ac.uk/9653/.
Full textAbstract:
Rab GTPases are known to define the vesicle trafficking pathways that underpin cell polarisation and migration. In this study it was demonstrated that Rab4, Rab11, Rab14 and the candidate Rab GDP-GTP exchange factor (GEF) FAM116A are all required for cell migration. This work also demonstrates that Rab14 and its GEF FAM116A localise to and act on an intermediate compartment within the transferrin recycling pathway acting upstream of Rab11 and downstream of Rab5. Further analysis revealed that a Rab14-dependent intermediate recycling compartment that is discrete from early and recycling endosomes, has an important function for cargo sorting in migrating cells. Cells depleted of Rab14 show increased levels of N-cadherin at junctional complexes and these cell-cell adhesions are unable to be resolved preventing cell migration. This results from decreased shedding of Ncadherin at the cell surface that is usually cleaved by A Disintegrin and Metalloprotease (ADAM) family protease ADAM10. In FAM116A- and Rab14-depleted cells, ADAM10 is trapped and therefore accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. Rab14 and its GEF FAM116A define an endocytic-recycling pathway that is essential for the trafficking of ADAM10 and therefore in turn regulate cell-cell junctions and cell migration.
31
Dunst, Sebastian. "Systematic characterization of Rab GTPase cell type expression and subcellular localization in Drosophila melanogaster." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-165234.
Full textAbstract:
The Rab family of small GTPases orchestrates intracellular endomembrane transport through the recruitment of diverse effector proteins. Since its first discovery in 1987, almost 70 Rab proteins have been identified in humans to date and their perturbed function is implicated in several hereditary and acquired diseases.
In this Ph.D. thesis, I systematically characterize cell type expression and subcellular localization of all Rab proteins present in Drosophila melanogaster utilizing a genetic resource that represents a major advance for studying membrane trafficking in vivo: the ’Drosophila YRab library’. This collection comprises 27 different D. melanogaster knock-in lines that harbor YFPMyc fusions to each Rab protein, referred to as YRab.
For each YRab, I present a comprehensive data set of quantitative and qualitative expression profiles across six larval and adult tissues that include 23 annotated cell types. The whole image data set, along with its annotations, is publicly accessible through the FLYtRAB database that links to CATMAID for online browsing of tissues.
I exploit this data set to address basic cell biological questions. i) How do differentiating cells reorganize their transport machinery to perform cell type-specific functions? My data indicates that qualitative and quantitative changes in YRab protein expression facilitate the functional specialization of differentiated cells. I show that about half of the YRab complement is ubiquitously expressed across D. melanogaster tissues, while others are missing from some cell types or reflect strongly restricted cell type expression, e.g. in the nervous system. I also depict that relative YRab expression levels change as cells differentiate. ii) Are specific Rab proteins dedicated to apical or basolateral protein transport in all epithelia? My data suggests that the endomembrane architecture reflects specific tasks performed by particular epithelial tissues, rather than a generalized apicobasal organization. I demonstrate that there is no single YRab that is similarly polarized in all epithelia. Rather, different epithelial tissues dynamically polarize the subcellular localization of many YRab compartments, producing membrane trafficking architectures that are tissue- and stage-specific.
I further discuss YRab cell type expression and subcellular localization in the context of Rab family evolution. I report that the conservation of YRab protein expression across D. melanogaster cell types reflects their evolutionary conservation in eukaryotes. In addition, my data supports the assumption that the flexible deployment of an expanded Rab family triggered cell differentiation in metazoans.
The FLYtRAB database and the ’Drosophila Rab Library’ are complementary resources that facilitate functional predictions based on YRab cell type expression and subcellular localization, and to subsequently test them by genetic loss-of-function experiments. I demonstrate the power of this approach by revealing new and redundant functions for Rab23 and Rab35 in wing vein patterning.
My data collectively highlight that in vivo studies of endomembrane transport pathways in different D. melanogaster cell types is a valuable approach to elucidate functions of Rab family proteins and their potential implications for human disease.
32
Johns, Helen Louise. "Regulation of FMDV infection by cellular rab GTPases." Thesis, University of Surrey, 2007. http://epubs.surrey.ac.uk/843907/.
Full textAbstract:
FMDV infection is initiated by virus binding to integrin receptors at the cell surface. Virus binding to the integrin triggers internalization of the virus-receptor complex which enters the cell via clathrin-dependent endocytosis. The low pH within endosomes triggers capsid disassembly and translocation of the viral RNA across the endosomal membrane into the cytosol. The precise identity of the endocytic compartment from which infection by FMDV takes place is currently unknown. Rab GTPases are central regulators of endocytosis. Each rab protein is enriched in one or more specific membrane compartments. In this study dominant-negative versions of a number of rabs are used to investigate the early events in FMDV infection of a pig kidney cell line (IBRS2). Infection is inhibited by expression of dominant-negative rab5 (which inhibits formation of early endosomes) but not by dominant-negative rab4 (which inhibits rapid recycling from early endosomes to the plasma membrane) or by dominant-negative rab9 (which inhibits trafficking from late endosomes to the Golgi). Dominant negative rab11, which inhibits a slower recycling pathway via recycling endosomes inhibits FMDV infection to an extent, although the effect of dominant-negative rab5 on infection is greater. While a dominant-negative form of rab7 (which regulates trafficking from early to late endosomes) unable to bind membranes inhibits FMDV infection, a membrane binding but inactive rab7 does not. This inhibition is shown to be at the stage of replication rather than entry. It is suggested that rab7 may be required for intracellular virus replication, possibly anchoring the replication complex to the replication vesicle.
33
Allin, Christoph. "Untersuchung der NF1-333-katalysierten GTPase von humanem H-ras p21 durch FT-IR-Spektroskopie." [S.l. : s.n.], 1999. http://deposit.ddb.de/cgi-bin/dokserv?idn=959152709.
Full text
34
Fransson, Åsa. "Cell signaling by Rho and Miro GTPases : Studies of Rho GTPases in Cytoskeletal Reorganizations and of Miro GTPases in Mitochondrial Dynamics." Doctoral thesis, Uppsala University, Ludwig Institute for Cancer Research, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8514.
Full textAbstract:
The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology.
A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought.
In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.
35
Sharma, Ankur. "Development of nanoparticulate drug delivery systems for anti-metastatic Ran GTPase therapeutics." Thesis, Ulster University, 2017. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.725342.
Full text
36
Gunasekharan, V. K. "Transcriptional Regulation of Human Ran GTPase and it's role in Malignant Transformation." Thesis, Queen's University Belfast, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.527707.
Full text
37
Tyler, Adam Michael. "Manipulating Rab GTPase activity in wheat to alter gluten quality for breadmaking." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12914/.
Full textAbstract:
In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialised vacuoles for the storage of protein. The important gluten proteins of wheat are transported to the protein bodies they are stored in by two distinct routes. One route consists of vesicles that bud directly off the ER, while the other involves transport through the Golgi (Arcalis et al, 2004). In plants, the RabD clade mediates ER to Golgi vesicle transport (Batoko et al, 2000). Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared in phenetic trees. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down-regulation by RNA interference (RNAi) and an RNAi construct was used to transform wheat plants. Using real time PCR, all four available RabD genes were shown to be knocked down in the developing endosperm of transgenic wheat. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE-HPLC found that a smaller proportion of HMW-GS and large LMW-GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS-PAGE was seen in the transgenic grain
38
Kawato, Mitsunori. "Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway." Kyoto University, 2008. http://hdl.handle.net/2433/135808.
Full text
39
Reynier, Marie. "Rôle des GTPases Rab dans le trafic des corps lamellaires épidermiques." Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30227.
Full textAbstract:
La couche cornée, couche la plus superficielle de l'épiderme, assure une fonction de barrière multifonctionnelle vitale pour l'organisme. Le maintien de cette barrière dépend de la fonctionnalité des cellules sous-jacentes, les kératinocytes granuleux, dernière couche de cellules vivantes de l'épiderme. Les kératinocytes granuleux contiennent dans leur cytoplasme de nombreux organites tubulo-vésiculaires sécrétoires, appelés corps lamellaires (CL). Les CL jouent un rôle majeur dans la formation et le maintien de cette barrière en déversant leur contenu (lipides, lipases, protéases, inhibiteurs de protéases, peptides antimicrobiens,...) à la transition couche cornée - couche granuleuse. Le trafic intracellulaire des CL est donc un processus déterminant dans l'homéostasie de la couche cornée. Cependant, les mécanismes moléculaires impliqués dans la régulation de ce trafic ne sont pas élucidés. Précédemment, au laboratoire, une analyse systématique par spectrométrie de masse des protéines associées aux CL a permis d'identifier plusieurs GTPases de la famille Rab et certains de leurs effecteurs. Ces protéines étant des régulateurs majeurs du trafic vésiculaire dans tous les types cellulaires, elles pourraient jouer un rôle dans le routage des CL et, ainsi, dans la fonctionnalité de la barrière épidermique. L'objectif de ma thèse était d'identifier les GTPases Rab et effecteurs impliqués dans la régulation du trafic intracellulaire des CL. Dans un premier temps, j'ai mis en évidence que la GTPase Rab11a est fortement exprimée dans les kératinocytes granuleux où elle est partiellement associée aux CL. J'ai montré que la déplétion de Rab11a dans un modèle tridimensionnel d'épiderme reconstruit in vitro réalisée grâce à la technique d'interférence à l'ARN, induit une diminution de la densité et de la sécrétion des CL. L'extinction de Rab11a entraîne également une baisse du taux de céramides et de cholestérol, une désorganisation des lipides intercornéocytaires et une augmentation de la perméabilité de la couche cornée. Dans les épidermes reconstruits déplétés, les composants des CL non-sécrétés sont adressés vers une voie de dégradation lysosomale. Dans un deuxième temps, j'ai observé que la perte d'expression de Rab11a induit une anomalie de distribution du moteur moléculaire Myosine-Vb, effecteur majeur de Rab11a. J'ai donc analysé les conséquences de la déplétion de Myosine-Vb dans les épidermes reconstruits et montré qu'elle induit un phénotype comparable à celui observé lors de la déplétion de Rab11a. L'ensemble de ces résultats suggèrent fortement que le complexe bipartite Rab11a-Myosine-Vb constitue un régulateur majeur de la biogenèse des CL et ainsi, de l'homéostasie de la barrière épidermique. Mon travail de thèse est la première étape du décryptage des voies moléculaires impliquées dans la biogenèse des CL et contribue à une meilleure compréhension du rôle du trafic intracellulaire dans la constitution de la barrière épidermique. Il pourrait permettre de caractériser les mécanismes physiopathologiques associés à un défaut de trafic des CLThe stratum corneum, the most superficial layer of the epidermis, provides a multifunctional protective barrier which is vital for the organism. The maintenance of this barrier is directly dependent on the underlying granular keratinocytes which are the last living cells in the epidermis. The granular keratinocytes contain in their cytoplasm numerous tubulovesicular secretory organelles called lamellar bodies (LB). LB play a major role in the establishment and the maintenance of the epidermal barrier by releasing their content (lipids, lipases, proteases, protease inhibitors, antimicrobial peptides,...) at the junction between the stratum corneum and the stratum granulosum. Because of LB importance in the maintenance of the stratum corneum homeostasis, the regulation of their trafficking deserves further study.Previously, in my laboratory, a proteomic characterization of LB by mass spectrometry has identified several Rab family GTPases and some of their effectors. In any cell type, from yeast to human, Rab GTPases are considered as major regulator of vesicular trafficking. Thus, I postulated that these proteins could play a role in the regulation of LB routing in the cytoplasm of granular keratinocytes. In this context, the aim of my thesis was to determine which Rab GTPases and effectors are involved in this process. In a first step, I demonstrated that Rab11a is strongly expressed in granular keratinocytes where it is associated with LB. I showed that Rab11a silencing using RNA interference technique in an in vitro tridimensional model of reconstructed human epidermis induces a decrease of LB density and secretion in granular keratinocytes. The Rab11a depletion also leads to a decrease of ceramide and cholesterol level and a disorganization of intercorneocyte lipids, generating a defective epidermal barrier. In depleted reconstructed epidermis, there is a missorting of non-secreted LB components, driven to the lysosomal degradation pathway. In a second step, I observed that Rab11a silencing affects distribution of its effector, the molecular motor Myosin-Vb. So, I analyzed the consequences of Myosin-Vb depletion in the model of reconstructed epidermis and I demonstrated that the phenotype obtained is similar that of a Rab11a depleted epidermis. Taken together, these results strongly suggest that the bipartite complex Rab11a-Myosin-Vb is able to regulate the biogenesis of LB in granular keratinocytes. Thus, this molecular complex is a crucial regulator of the epidermal barrier homeostasis. My thesis work is a first step in the deciphering of the molecular pathway involved in LB biogenesis. It is a breakthrough in the comprehension that membrane dynamic in the granular keratinocytes is a major regulator of epidermal barrier. It may contribute to a better understanding of pathophysiological mechanisms related to dysregulated LB trafficking in skin diseases
40
Reynolds, Jon. "Characterisation of the inositol 1,3,4,5- tetrakisphosphate-binding GTPase-activating protein, GAP1'I'P'4'B'P." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310690.
Full text
41
Gomes, Anita Raquel Quintal. "Studies on membrane-association and targeting of RAB GTPases." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396315.
Full text
42
Newbury, Lucy Jade. "Targeting Ras GTPases in murine models of renal fibrosis." Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/targeting-ras-gtpases-in-murine-models-of-renal-fibrosis(01debcfb-6d17-4452-b142-e686eb9d059e).html.
Full textAbstract:
End stage kidney disease (ESKD) affects over 5300 people. The progression of chronic kidney disease (CKD) to ESKD is characterised by cytokine stimulation, leading to the activation of myofibroblasts, resulting in fibrosis. Kirsten rat sarcoma (KNRas) has a key role in the proliferation of renal fibroblasts in vitro and has been highlighted as a possible target for fibrosis. Previous research in our laboratory showed that inhibiting KNRas in the UUO model inhibited renal fibrosis. The aim of this thesis was to investigate the outcome of inhibiting KNRas using Antisense Oligonucleotide (ASO) in the novel Chronic Folic Acid Nephropathy (CFAN) model to study the effect on both fibrosis and renal function. The effect of inhibiting KNRas was also investigated in vitro, to try and elucidate the mechanism by which KNRas controls the progression of fibrosis. KNRas knockdown with ASO143 resulted in 50% reduction in KNRas mRNA which was associated with: 37%-50% reduction in total collagen and protection of renal function (BUN) in the 12 week CFAN model. TGFβ1 treated cells showed an upN regulation i KNRas, Jag1 and Collagen 1a mRNA. Treatment with KNRas ASO was associated with a 3.5 fold reduction in Jag 1 and a 55% reduction in collagen 1a. Jag 1 has been linked to the progression of renal fibrosis via biNdirectional signalling with Notch 1. In conclusion, ASO knockdown of KNRas inhibits fibrosis in vitro and in vivo, and in some instances protects renal function. These results support the hypothesis that KNRas targeting may be beneficial in the treatment of renal fibrosis. Further work is required to further understand the relationship between Jag 1,Notch 1 and KNRas, but this data suggests that KNRas affects renal fibrosis in a Jag 1 dependent pathway.
43
Klähn, Marco. "Untersuchung des intrinsischen GTPase-Mechanismus von Ras p21 mit Mitteln der Molekulardynamik-Simulation und QM/MM-Kraftfeld." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969939345.
Full text
44
Baï, Siau Wei. "Caractérisation fonctionnelle du complexe Nup107 : un sous-complexe protéique du pore nucléaire chez la levure Schizosaccharomyces pombe." Paris 11, 2004. http://www.theses.fr/2004PA112251.
Full textAbstract:
Chez les eucaryotes, les pores nucléaires (NPC) contrôlent tous les échanges de macromolécules entre le noyau et le cytoplasme. Les caractéristiques générales de la structure du NPC et ses principaux constituants (appelés nucléoporines ou Nups) ont été conservés au cours de l'Evolution Contrairement au NPC de S. Cerevisiae et des Vertébrés, il existe actuellement peu de données sur le NPC chez S. Pombe. Par homologie de séquence avec les Nups du complexe Nup84 de S. Cerevisiae, j'ai nouvellement identifié le complexe Nup107 chez S. Pombe. En dépit des différences de viabilité des souches délétées pour ces Nups entre ces deux levures, l'analyse phénotypique des souches mutantes suggère que l'implication du complexe Nup84/107, dans la distribution des NPC au sein de l'enveloppe nucléaire ainsi que dans l'export nucléaire des ARNm, a été conservée au cours de l'Evolution. Par ailleurs, certaines des souches S. Pombe présentent des défauts de mitoses (condensation anormale de la chromatine, défauts de ségrégation des chromosomes et de cytocinèse/septation), suggérant un rôle additionnel du complexe Nup107 en mitose. Mes travaux ont montré l'existence d'une interaction génétique entre le complexe Nup107 et la petite GTPase Ran, suggérant que l'implication du complexe Nup107 dans les mécanismes de mitose pourrait être liée aux fonctions de Ran en mitose. Ainsi bien que le complexe Nup107 chez S. Pombe ne soit pas localisé aux kinétochores comme cela a été montré chez l'humain, mes travaux mettent en évidence la participation du complexe Nup84/107 au processus de mitose et la conservation au cours de l'Evolution du lien étroit entre le complexe Nup84/107 et RanThe overall organization of the nuclear pore complexes (NPC) has been evolutionary conserved. Yet variations in organisms likely provide important clues to NPC functions. While most NPC proteins (nucleoporins or Nups) have been identified in S. Cerevisiae and vertebrates, few nucleoporins have been characterized in the yeast Schizosaccharomyces pombe. During my PhD, through sequence homology with S. Cerevisiae, I have identified and further functionally characterized 6 novel S. Pombe nucleoporins. Biochemical approaches have confirmed the organization of these Nups within conserved NPC subcomplexes. While examination of the S. Pombe deletion mutants revealed different viability phenotype as compared to their S. Cerevisiae orthologues, functional studies indicated that the involvement of this complex in NPC distribution and mRNA export has been partly conserved between these highly divergent yeasts. Unexpectedly microscopic analyses of the S. Pombe mutants showed additional cell division defects (spindles, chromosome segregation and septation) reminiscent to some GTPase Ran-cycle mutants. Consistently I further showed a specific functional link between the SpNup107 complex and the Ran pathway. During mitosis, the Human Nup107 complex colocalizes with kinetochores suggesting an as yet unidentified evolutionary conserved role of these Nups in mitosis, even though this feature does not seem to be conserved in S. Pombe. These differences between yeast S. Pombe and vertebrates could be associated to some specificity related to closed versus opened mitosis. My work has thus provided additional clues of the involvement of the Nup107 complex in cell division mechanisms
45
Schatz, Christoph. "The role of the small GTPase ran during assembly of a mitotic spindle." [S.l.] : [s.n.], 2003. http://www.diss.fu-berlin.de/2003/262/index.html.
Full text
46
Loumrhari, Fatine. "Investigation of Rab GTPase interaction with focal adhesion proteins in breast cancer cells." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86771.
Full textAbstract:
The acquisition of cancer cell invasive properties is a critical early event in primary cancer progression to metastasis. Increasing experimental and clinical evidence supports that metastasis formation is initiated by the acquisition of cancer cell motile properties driven by extensive remodelling of the cell cytoskeleton and dynamic recycling of focal adhesion proteins (FA); the later link the extracellular matrix to the cell cytoskeleton and intracellular signaling. Cancer cell invasion has been associated with elevated expression/activation of several proteins, including members of Rab GTPases. These proteins are key regulators of membrane trafficking of proteins and membrane receptor recycling, including that of integrins. Elevated expression of Rab GTPases has been linked to increased cell migration and invasion. Previously, biochemical and proteomic studies from my host laboratory, using paired non-invasive and invasive breast cancer cells have revealed a rapid turnover of FA proteins in invasive compared to non-invasive cancer cells, as well as differential expression of several Rab GTPase members, including Rab5, Rab11 and Rab7. Thus, I tested the hypothesis that Rab GTPases may regulate FA protein turnover, in part via interaction with focal adhesion kinase (FAK), a central protein of master focal adhesion signalling. Immunofluorescence and immunoprecipitation assays reveal that Rab11 and FAK interact and colocalize at FA sites. Development of cell lines where Rabs are depleted using siRNA revealed an impact of Rab on cell migration; Rab inhibition inhibited cell migration. I propose that Rab GTPases, and in particular Rab11 may contribute to the regulation of FAK function in FA turnover and cell migration.Le cancer invasif du sein reste une maladie extrêmement courante et une cause majeure de mortalité parmi les personnes atteintes du cancer. Un événement par lequel les cellules du cancer se propagent est initié par l'acquisition de propriétés mobiles conduites par le remodellement du cytosquelette cellulaire et par le roulement dynamique de protéines d'adhérence focale (FA); relient la matrice extracellulaire au cytosquelette cellulaire. Le cancer invasif du sein a été associé avec l'expression élevée de certaines proteines surnommées les Rab GTPases. Ils sont connus comme des regulateurs essentiels dans la circulation membraneuse y compris le recyclage des recepteurs incluant celles des integrins, protéines d'adhérence focale qui agissent en tant que mediateur pour l'adherence des cellules a la matrice extracellulaire. Leur expression élevée, a en fait, favorisé la migration et l'invasion des cellules, contribuable a la métastase du cancer. Auparavant, des études "proteomiques" sur des cellules non-invasives contre des cellules invasives du cancer du sein ont révélé un roulement rapide de protéines d'adhérence focales (FA) dans les cellules invasives, ainsi q'une expression différentielle de plusieurs membres de Rab GTPases, y compris Rab5, Rab11 et Rab7. Ainsi, nous testons l'hypothèse que Rab GTPase interagit avec la protein kinase d'adhérence focale (FAK), qui sert comme mediateur pour les voies de transduction des signaux aux sites d'adherence focale. Nous supposons aussi que les Rabs pourraient jouer un role important dans la regulations de la circulation de FA dans le cancer invasive du sein. Les experiences faites avec l'immunofluorecence et l'immunoprecipitation révèlent que Rab11 et FAK interagissent et colocalisent ensembles. Le développement de lignes de cellules où les Rabs ont été diminués en utilisent le siRNA démontre son effet sur la migration de cellules. Je propose que Rab11 est un élément decisif qui po
47
Kocher, Hemant Mahendrakuma. "Ras GTPase isoforms in normal and pathological human and animal kidney and pancreas." Thesis, King's College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420100.
Full text
48
Kirchhelle, Charlotte. "RAB-A5c GTPase and the regulation of cell geometry during Arabidopsis root organogenesis." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:a2a1279e-32fd-487b-8f15-5b350e7a2bf4.
Full textAbstract:
A fundamental challenge in biology is to explain how different organisms develop the intricate anatomical forms observed in nature. During morphogenesis, biochemical, mechanical, and geometric information has to be integrated across multiple organisational scales, from multicellular organs over individual cells down to subcellular components. In multicellular plants, individual cells are fixed in their relative position in the tissue by their surrounding rigid cell wall. Therefore, plant organogenesis requires control over division plane orientation and anisotropic cell wall growth, which each require spatial patterning of cells. Polyhedral plant cells can display complex patterning in which individual faces are established as biochemically distinct domains by endomembrane trafficking. This thesis shows that the specification of an additional cellular spatial domain by the endomembrane system, a cell's geometric edges, is crucial for lateral root organogenesis. Membrane vesicles lying immediately beneath the plasma membrane at cell edges were revealed through localisation of RAB-A5c, a plant GTPase of the Rab family of membrane-trafficking regulators. RAB-A5c compartments were enriched at growing edges, and associated with the cortical microtubule array. Specific inhibition of RAB-A5c activity grossly perturbed cell geometry and organ growth in developing lateral organs. Quantitative 4D cell growth analysis revealed RAB-A5c inhibition indedependently caused perturbations of cell growth anisotropy and cytokinesis without disrupting default membrane trafficking. To investigate the underlying mechanism of RAB-A5c-mediated growth regulation, hypotheses for RAB-A5c function in modulating cortical microtubule array orientation and cell wall mechanical properties were tested. Based on a combination of quantitative image analysis, pharmacological and genetic approaches, and mechanical modelling, RAB-A5c is proposed to locally modify cell wall properties during growth, and two possible models for RAB-A5c function and their implications for cell-, tissue- and organ-level growth control are discussed.
49
Attia, Mehdi. "Implication de la GTPase RagA dans l’activation et la polarisation de la réponse lymphocytaire T." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30214.
Full textAbstract:
Les lymphocytes T (LT) jouent un rôle clé dans la mise en place d’une réponse immunitaire efficace. De part des besoins énergétiques et biosynthétiques distincts, leur activation, leur prolifération et leur différenciation fonctionnelle requièrent un contrôle métabolique très fin. La kinase mTOR est apparue comme un régulateur important de la biologie des LT. En effet, cette kinase contrôle le métabolisme et permet une augmentation de la synthèse d’énergie notamment par une augmentation de la glycolyse, indispensable à l’activation des LT. mTOR détecte la disponibilité des nutriments (ex. les acides aminés et le glucose) ainsi que les facteurs de croissance, puis intègre de tels signaux pour réguler le métabolisme des LT. Un rôle clé des acides aminés dans la réponse des LT a été mis en évidence. La petite protéine-G RagA joue un rôle clé dans la détection du glucose et des acides aminés ramifiés, requise pour l’activation de mTOR dans le complexe mTORC1, impliqué dans le contrôle du métabolisme. Afin d’appréhender l’influence du microenvironnement métabolique sur l’activation, la prolifération et la polarisation des LT auxiliaires, nous avons généré et analysé des souris avec des mutations de RagA dans les LT. Les souris mutantes ne présentent aucun signe macroscopique de perturbations du système immunitaire tel que des pathologies autoimmunes ou le développement de tumeurs. Le développement des LT dans le thymus est globalement normal, même si l’on peut observer une légère diminution du développement des LT régulateurs. En périphérie, l’homéostasie immunitaire ne semble pas altérée mis à part une légère diminution du pourcentage de LT mémoires. Nous avons constaté que la perte de RagA entraîne une diminution substantielle de l’activité de mTORC1 observée après activation des LT mais, de façon inattendue, pas une abolition totale. A l’inverse, nous avons observé une augmentation de l’activité de mTORC2 dans les cellules KO. De façon plus surprenante, nous avons mis en évidence que, très rapidement après la délétion de RagA dans le thymus, une faible activité basale de mTORC1 se met en place. Précocement après activation, les LT RagA KO ne présentent pas de problème de survie, cependant ils prolifèrent moins rapidement, ce qui est vraisemblablement dû à un apport d’énergie plus faible par glycolyse. Nous avons constaté que des LT RagA-KO activés in vitro dans des conditions « neutres » expriment spontanément des niveaux plus élevés de T-bet, facteur de transcription « maître » des lymphocytes T auxiliaires de type I (Th1). Aussi suite à une activation des LT en condition polarisante Th1, nous avons observé davantage de cellules RagA-KO que WT produisant de l’interféron-γ. Ces résultats montrent que l’activité de RagA, et par conséquent vraisemblablement de mTORC1, inhibe la différenciation Th1. Nous avons pu constater que les LT RagA-KO favorisent la différenciation Th1 au moins en partie par des mécanismes intrinsèques et extrinsèques. De plus, nous observons une activité tardive de mTORC1 dans les LT RagA-KO. Nous émettons l’hypothèse que RagA inhibe l’activité tardive de mTORC1 et que cette activité tardive permet une meilleure différenciation en Th1. En conclusion, nos résultats montrent que l’absence de la GTPase RagA dans les LT diminue l’activité de mTORC1 sans l’abolir totalement. De façon importante et surprenante, nous démontrons que malgré la baisse d’activité de mTORC1 en absence de RagA, la différenciation en lymphocytes Th1 est augmentée. Ainsi, la GTPase RagA semble avoir un rôle inhibiteur de la différenciation en Th1 potentiellement en inhibant une activité à long terme de mTORC1T lymphocytes play a key role in the development of an effective immune response. Because of their distinct energy and biosynthetic needs, their activation, proliferation and functional differentiation require very fine metabolic control. The mTOR kinase has emerged as an important regulator of the biology of helper T cells. Indeed, this kinase controls the metabolism and allows an increase in the synthesis of energy in particular by an increase in glycolysis, essential for the activation of T cells. mTOR detects the availability of nutrients, such as amino acids, glucose and growth factors, and then integrates such signals to regulate T cell metabolism. Studies have shown a key role of amino acids in the response of T cells. The small RagA-G protein plays a key role in the detection of glucose and branched amino acids required for the activation of mTOR in the mTORC1 complex involved in metabolic control. In order to understand the influence of the metabolic microenvironment on the activation, proliferation and polarization of helper T cells we generated and analyzed mice with mutations of RagA in T cells. Mutant mice show no signs of immune system disturbances such as autoimmune pathologies or tumor development. T cell development in the thymus is g normal even though a slight decrease in the development of regulatory T cells can be observed. In the periphery, immune homeostasis does not seem to be altered except for a slight decrease in the percentage of memory T cells.We found that the loss of RagA results in a substantial decrease in mTORC1 activity after T cell activation but unexpectedly not complete abolition. Conversely, we observed an increase in mTORC2 activity in KO cells. More surprisingly, we have shown that, very soon after the deletion of RagA in the thymus, a low basal activity of mTORC1 takes place. Early after activation, RagA KO T cells did not present a survival problem, however they proliferated less rapidly, which is probably due to a lower energy intake by glycolysis. We have found that RagA KO T cells activated in vitro under "neutral" conditions spontaneously express higher levels of T-bet, the "master regulator" transcription factor of type I (Th1) helper T cells. Therefore, following activation of T cells in polarizing condition Th1, we observed more RagA KO cells than wt producing interferon-γ. These results show that the activity of RagA, and therefore presumably of mTORC1, inhibits Th1 differentiation. We have seen that RagA KO cells favor Th1 differentiation by intrinsic and extrinsic mechanisms. We hypothesize that IFN-γ, more produced by RagA-KO cells, is involved. In addition, we observed a late activity of mTORC1 in RagA-KO LT. We hypothesize that RagA inhibits the late activity of mTORC1 and that this late activity allows better Th1 differentiation. In conclusion, our results show that the absence of RagA GTPase in T cells decreases the activity of mTORC1 without completely abolishing it. Significantly and surprisingly, we demonstrate that despite the decrease in mTORC1 activity, Th1 cell differentiation is increased in the absence of RagA. Thus, RagA GTPase appears to have an inhibitory role in Th1 differentiation potentially by inhibiting a long-term activity of mTORC1
50
Qi, Xingyun. "Function and regulation of small RAB-A1 GTPases in Arabidopsis." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110604.
Full textAbstract:
In this study, the function and regulation of Rab-A1 proteins are investigated in the model plant species Arabidopsis. Rab proteins are a family of small Ras-like GTPases that regulate the vesicle transport during membrane trafficking. The subfamily of Rab-A proteins, homologues of Ypt31/32 in yeast and Rab11 in animals, is greatly elaborated in plants. In Arabidopsis, 26 out of 57 putative Rab proteins are Rab-A members and they can be further categorized into 6 subclasses from Rab-A1 to Rab-A6. The unique radiation of the Rab-A subfamily in the plant lineage is thought to be a mechanism to meet the dynamic membrane trafficking around the trans-Golgi network (TGN) in plant cells. With fluorescence based imaging, I revealed that, RAB-A1c, a member of the Rab-A1 subclass, is strongly co-localized with members of the Rab-A2 and Rab-A4 subclasses in a population of TGN that only partially overlaps with VHA-a1-mRFP, a TGN marker. RAB-A1c is relocated to the growing cell plate in mitotic cells. Interestingly, this RAB-A1c positive compartment and the relocation of RAB-A1c to the cell plate in mitotic cells are sensitive to endosidin1 (ES1), a drug recently identified as an actin stabilizer that selectively interrupts recycling of several plasma membrane proteins from the TGN. Furthermore, although there is likely functional redundancy in Rab-A1 members, the root growth of rab-a1a/b/c triple mutant is slightly retarded and, importantly, it displays hypersensitivity to ES1. Histological staining revealed that enhanced defect in cytokinesis contributes to ES1 hypersensitivity in root growth of the triple mutant. Thus, I conclude that Rab-A1 proteins are involved in cytokinesis and they act in a membrane trafficking pathway(s) that is sensitive to ES1.As the molecular switch of membrane trafficking, Rab proteins are under the regulation of the guanine exchange factor (GEF). In yeast, TRAPPII (transport protein particle II), a ten-subunit protein complex (seven subunits shared with TRAPPI including Trs20, Trs23, Trs31, Trs33, Trs85, Bet3 and Bet5, and three TRAPPII specific subunits including Trs65, Trs120 and Trs130) has been reported as a GEF for Ypt31/32. Using a combined approach of in-vivo imaging and genetics, we revealed that mutations in AtTrs120 and AtTrs130, homologues of two TRAPPII-specific subunits in Arabidopsis, cause defect in cytokinesis as well as in cell polarity. In the background of attrs120 and attrs130, vesicles and tubular-vesicular structures are abnormally accumulated, but ER-Golgi transport, trafficking route to the vacuole and endocytosis appear normal. Using secGFP as a secretion marker, I revealed that transport of secGFP can be inhibited at the TGN. In addition, recycling of PIN2 and AUX1, but not PIN1 is also impaired in attrs120 or attrs130. I found that a functional YFP fused AtTrs130 co-localizes with GFP-RAB-A1c at a population of TGN that is sensitive to ES1. In attrs130, however, the majority of GFP-RAB-A1c is delocalized to the cytosol. Furthermore, the constitutive active RAB-A1c(Q72L), but not RAB-D2a(Q67L), could partially rescue attrs130 in root growth. Taken together, I conclude that TRAPPII in Arabidopsis may function upstream of RAB-A1c, possibly as a GEF, in post-Golgi membrane trafficking.Au cours de cette étude, je me suis intéressée à la fonction et à la régulation des protéines Rab-A1 au cours de la division cellulaire chez Arabidopsis.Parmis la famille de petites GTPases Ras-like, la sous-famille des protéines Rab régulent le transport vésiculaire durant le trafic membranaire. La sous-famille Rab-A, homologue de Ypt31/32 chez la levure et de Rab11 chez les animaux, est considérablement sophistiquée chez les plantes. Chez Arabidopsis, 26 des 57 protéines Rab prédites sont des membres de la famille des protéines Rab-A et peuvent donc êtres organisés en 6 sous-classes, de Rab-A1 à Rab-A6. Cette arborisation unique des protéines Rab-A chez les plantes suggère un mécanisme spécifique lié au dynamisme du trafic membranaire autour du réseau Trans-golgien (Trans-Golgi Network ou TGN). Rab-A1c strictement avec certain membres des familles Rab-A2 et Rab-A4, mais ne co-localise que partiellement avec VHA-a1-mRFP, marqueur du TGN, suggérant une restriction de cette protéine à une sous-population du TGN. Dans les cellules mitotiques, RAB-A1c est relocalisée au niveau de la plaque cellulaire en croissance. De manière intéressante, ce compartiment ainsi que la relocalisation de RAB-A1c à la plaque cellulaire pendant la mitose sont sensibles à l'endosidine 1 (ES1), un stabilisateur d'actine récemment identifié qui interrompt de façon sélective le recyclage de différentes protéines de la membrane cellulaire du TGN. De plus, malgré la redondance fonctionnelle entre les membres de la famille Rab-A1, les racines présentent un défaut de croissance dans les triples mutants rab-a1a/b/c, démontrant une hypersensibilité à ES1. Ce retard de croissance, causé par un défaut de mitose, contribue à l'hypersensibilité à ES1 dans les racines en formation. Finalement, les protéines Rab-A1 seraient donc sensibles à ES1 durant la cytokinese et joueraient un rôle important dans la régulation du trafic membranaire.Les protéines Rab, en tant que déclencheur moléculaire du trafic membranaire, sont régulées par les facteurs d'échange de guanine (GEFs). Chez la levure, TRAPII, un complexe protéique de 10 sous-unités (7 sous-unités partagées avec TRAPI incluant Trs20, Trs23, Trs31, Trs33, Trs85, Bet3 et Bet5, et 3 sous-unités spécifiques à TRAPII, appelées Trs65, Trs120 et Trs130) a été identifié comme étant le facteur GEF pour YPT31/32. En utilisant une approche combinant l'imagerie in vivo et la génétique, nous avons révélé que les mutants attrs120 et attrs130, codant deux des sous-unités spécifiques de TRAPII chez Arabidopsis, présentent des défauts de cytokinese semblable à une absence de polarité cellulaire. Dans ces mutants, les vésicules ainsi que les structures vésicule-tubules s'accumulent anormalement. Toutefois, le transport entre le Reticulum endoplasmique (RE) et le Golgi semble être normal. En utilisant secGFP comme marqueur de la sécrétion, j'ai démontré que le transport de cette protéine vers le TGN est inhibé dans les mutants attrs120 et attrs130. De plus, le recyclage de PIN2 et AUX1, mais pas celui de PIN1 est ralenti dans ce contexte mutant. J'ai démontré que AtTrs130-YFP co-localise parfaitement avec GFP-RAB-A1c dans la sous-population de TGN sensible à ES1. Cependant, dans les mutants attrs130, la majorité des protéines GFP-RAB-A1c est délocalisée vers cytoplasme. Dans les mutants attrs130, la surexpression de RAB-A1c(Q72L), constitutionnellement active, compense partiellement le phénotype de défaut de croissance des racines, contrairement à celle de RAB-D2a(Q67L). L'ensemble de ces résultats montre que TRAPII, chez Arabidopsis, pourrait être le facteur GEF en amont des protéines Rab-A1 dans le trafic membranaire post-golgien.