Tesis:

1

ZAMBERLAN, MARGHERITA. "La piccola GTPasi Rap1 interposta tra la proteina mitocondriale Opa1 e l'inibizione dell'angiogenesi". Doctoral thesis, Università degli studi di Padova, 2022. https://hdl.handle.net/11577/3460979.

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OPA1 is a protein with pleiotropic functions ranging from the orchestration of mitochondrial fusion and cristae remodeling to transcriptional reprogramming 1, 2. Here we present two different mechanisms by which OPA1 exerts a transcriptional regulation activity in endothelial and breast cancer cells. Mitochondria are dynamic organelles that are now recognized as regulators of signal transduction able to impact on cellular genetic programs 3, 4. Increasing evidence support a fundamental role for mitochondrial shape in the orchestration of cellular transcriptional programs, but how cells sense and respond to changes in mitochondrial shape is unclear 5. We recently discovered that angiogenesis is transcriptionally modulated by the key mitochondrial fusion gene OPA1 through NFκB activation1. In particular, ablation of OPA1 in vivo and in vitro leads to developmental and tumor angiogenesis inhibition 1. A deep RNA sequencing analysis identified a signature for the Ras-proximate-1 RAP1, and its cyclic AMP (cAMP)-activated nucleotide exchange factor EPAC1 upon OPA1 deletion in Human Umbilical Vein Endothelial Cells (HUVECs). Previously, several studies had reported the essential role of Rap1 in developmental angiogenesis and vessel stabilization 6, 7. After birth, Rap1 is not essential, but it participates in the maintenance of vasculature and nitric oxide (NO) homeostasis 6, 8. Albeit EPAC1 is highly abundant and cover numerous functions in endothelial cells, its role in angiogenesis remained to be clarified 9. Likewise, EPAC1 was shown to be important for endothelial cells biology 10, 11. A handful of studies retrieved EPAC1 in mitochondria, and RAP1 in mitochondria associated membranes (MAMs) by proteomics, suggesting that they might be linked to mitochondria 12, 13. Whether the EPAC1/RAP1 axis could sense changes in mitochondria driven by OPA1 deletion was unknown. Our results show that EPAC1 and RAP1 localize in proximity to mitochondria and in MAMs, that are emerging as hubs for mitochondria-derived signals. Moreover, EPAC1 accumulates on mitochondria upon pharmacological activation and following OPA1 silencing. OPA1 silencing results in an increase in Ca2+ and in localized cAMP increase in proximity of mitochondria that in turn activated EPAC1 and therefore RAP1. Notably, Ca2+ chelation by BAPTA-AM treatment suppress the EPAC1/RAP1 activation elicited by OPA1 downregulation. Furthermore, the analysis of angiogenic parameters like migration and tubulogenesis revealed that the blockage of EPAC1 and RAP1 signaling could correct the defects caused by OPA1 downregulation. Thus, our work places EPAC1/RAP1 in the retrograde signaling pathway connecting mitochondria to angiogenesis and highlights the intricate network of signals and second messengers that can execute transcriptional changes when mitochondria are perturbed.
OPA1 is a protein with pleiotropic functions ranging from the orchestration of mitochondrial fusion and cristae remodeling to transcriptional reprogramming 1, 2. Here we present two different mechanisms by which OPA1 exerts a transcriptional regulation activity in endothelial and breast cancer cells. Mitochondria are dynamic organelles that are now recognized as regulators of signal transduction able to impact on cellular genetic programs 3, 4. Increasing evidence support a fundamental role for mitochondrial shape in the orchestration of cellular transcriptional programs, but how cells sense and respond to changes in mitochondrial shape is unclear 5. We recently discovered that angiogenesis is transcriptionally modulated by the key mitochondrial fusion gene OPA1 through NFκB activation1. In particular, ablation of OPA1 in vivo and in vitro leads to developmental and tumor angiogenesis inhibition 1. A deep RNA sequencing analysis identified a signature for the Ras-proximate-1 RAP1, and its cyclic AMP (cAMP)-activated nucleotide exchange factor EPAC1 upon OPA1 deletion in Human Umbilical Vein Endothelial Cells (HUVECs). Previously, several studies had reported the essential role of Rap1 in developmental angiogenesis and vessel stabilization 6, 7. After birth, Rap1 is not essential, but it participates in the maintenance of vasculature and nitric oxide (NO) homeostasis 6, 8. Albeit EPAC1 is highly abundant and cover numerous functions in endothelial cells, its role in angiogenesis remained to be clarified 9. Likewise, EPAC1 was shown to be important for endothelial cells biology 10, 11. A handful of studies retrieved EPAC1 in mitochondria, and RAP1 in mitochondria associated membranes (MAMs) by proteomics, suggesting that they might be linked to mitochondria 12, 13. Whether the EPAC1/RAP1 axis could sense changes in mitochondria driven by OPA1 deletion was unknown. Our results show that EPAC1 and RAP1 localize in proximity to mitochondria and in MAMs, that are emerging as hubs for mitochondria-derived signals. Moreover, EPAC1 accumulates on mitochondria upon pharmacological activation and following OPA1 silencing. OPA1 silencing results in an increase in Ca2+ and in localized cAMP increase in proximity of mitochondria that in turn activated EPAC1 and therefore RAP1. Notably, Ca2+ chelation by BAPTA-AM treatment suppress the EPAC1/RAP1 activation elicited by OPA1 downregulation. Furthermore, the analysis of angiogenic parameters like migration and tubulogenesis revealed that the blockage of EPAC1 and RAP1 signaling could correct the defects caused by OPA1 downregulation. Thus, our work places EPAC1/RAP1 in the retrograde signaling pathway connecting mitochondria to angiogenesis and highlights the intricate network of signals and second messengers that can execute transcriptional changes when mitochondria are perturbed.

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2

Normandin, Caroline. "Identification et caractérisation de GTPases Activating Proteins spécifiques à la petite GTPase RAB21". Mémoire, Université de Sherbrooke, 2017. http://hdl.handle.net/11143/11544.

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L’autophagie est un processus de dégradation et de recyclage des composés cellulaires. Ce mécanisme est nécessaire que ce soit à l’état basal pour éliminer des agrégats protéiques ou des organites endommagés ou en condition de stress, tels que la carence nutritionnelle, l’hypoxie ou encore des traitements anticancéreux. De ce fait, l’autophagie est un processus essentiel à la survie ainsi qu’au maintien de l’homéostasie cellulaire. Connaître les joueurs et comprendre les mécanismes de régulation de l’autophagie sont donc importants. Les GTPases RABs sont des régulateurs importants de ce processus. Celles-ci agissent comme des interrupteurs moléculaires permettant d’exécuter rapidement des fonctions dans la cellule. Les RABs sont activées par des Guanine Nucleotide Exchange Factors (GEF) alors que les GTPase Activating Proteins (GAP) accélèrent la désactivation de la RAB. RAB21 est essentielle dans les étapes tardives de l’autophagie. En effet, RAB21 est activée par la carence nutritionnelle, via sa GEF MTMTR13, et permet le trafic d’une SNARE requise pour le flux autophagique. Lors d’une carence prolongée, l’activité de RAB21 diminue rapidement, suggérant ainsi le rôle d’une GAP dans cette régulation négative. Toutefois, aucune GAP pour RAB21 n’a été identifiée jusqu’à maintenant. Un criblage génétique chez la drosophile a permis d’identifier quelques candidats. Suite à des essais d’interactions protéiques, il s’est avéré que seule la GAP TBC1D25 interagissait avec RAB21. De plus, cette interaction est augmentée en fonction de la carence nutritionnelle. Des immunofluorescences par microscopie confocale ont révélé que l’interaction RAB21-TBC1D25 était située en partie au niveau des endosomes précoces. Par ailleurs, une activation prolongée de RAB5, située sur les endosomes précoces, inhibe l’interaction RAB21-TBC1D25. De plus amples expériences devront être réalisées afin d’expliquer ces résultats. Dans un autre ordre d’idée, RAB21 est surexprimée dans les cellules ayant un flux autophagique élevé ainsi que dans certaines tumeurs de cancer du côlon (données non publiées du laboratoire). L’expression de Tbc1d25 dans ces mêmes tumeurs ne semble pas augmentée, indiquant que TBC1D25 pourrait être un inhibiteur autophagique spécifique aux cellules ayant un flux autophagique élevé. À la lumière des résultats obtenus, TBC1D25 semble être une GAP pour RAB21 qui permet sa régulation négative suivant l’activation de l’autophagie induite par la carence nutritionnelle.
Abstract : Autophagy is defined as the lysosomal degradation and recycling of cellular constituents. At basal levels, autophagy eliminates protein aggregates or damaged organelles. In condition of stress, such as in condition of nutritional deficiency, hypoxia or cancer treatments, autophagy allow cells to adapt and survive. Therefore, autophagy is an essential system required for survival and maintenance of cellular homeostasis. It is thus essential to identify the cellular entities and mechanisms regulating this process. RAB GTPases were identified as master regulators of autophagy. These particular proteins act as molecular switches for the rapid execution of cellular responses. RABs are activated by Guanine Nucleotide Exchange Factors (GEF) whereas GTPase Activating Proteins (GAP) accelerates RAB deactivation. RAB21 is essential in the late stages of autophagy. Indeed, RAB21 is activated by nutritional deficiency, via its GEF MTMTR13, to allow trafficking of a SNARE required for autophagic flux. During starvation, RAB21 is deactivated which suggest that a GAP could negatively regulate RAB21 activity. However, to date no GAP for RAB21 has been identified. An eye modifier genetic screen in Drosophila was performed to identify potential RAB21 GAPs and some candidates were identified. As a result of this screen, the GAP TBC1D25 was identified as interacting with RAB21. Moreover, this interaction was increased by starvation. Proximity ligation assays revealed that the RAB21-TBC1D25 interaction partially localized at early endosomes. Moreover, prolonged activation of RAB5, located at early endosomes, inhibited RAB21-TBC1D25 interaction. Further experiments will be carried out to explain these results. With respect to the roles of autophagy in cancer, RAB21 was shown to be overexpressed in cells with high autophagic flux as well as in some colon cancer tumors. Importantly, the expression of Tbc1d25 in these same tumors does not appear to be increased, indicating that TBC1D25 could be an autophagic inhibitor specific to cells with a high autophagic flow. My work suggests that TBC1D25 could function as a GAP to negatively regulate RAB21 activity in condition of prolonged starvation.

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3

Chan, King-chung Fred y 陳敬忠. "Functional characterization of StAR-related lipid transfer domain containing 13 (DLC 2) RhoGAP in the nervous system". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43278449.

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4

Chan, King-chung Fred. "Functional characterization of StAR-related lipid transfer domain containing 13 (DLC 2) RhoGAP in the nervous system". Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43278449.

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5

Paul, Florian [Verfasser]. "Developing quantitative GTPase affinity purification (qGAP) to identify interaction partners of Rho GTPases / Florian Paul". Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1069532711/34.

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6

Paul, Florian Ernst Rudolf Benjamin [Verfasser]. "Developing quantitative GTPase affinity purification (qGAP) to identify interaction partners of Rho GTPases / Florian Paul". Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1069532711/34.

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7

Bery, Nicolas. "Nouvelle stratégie de ciblage de la GTPase RhoB : développement d'intracorps conformationnels sélectifs et leur fonctionnalisation en tant qu'inhibiteurs intracellulaires de l'activité de RhoB". Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2734/.

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La GTPase RhoB partage 85% d'homologie avec RhoA et RhoC. Ces protéines alternent entre deux conformations : une active liée au GTP et une inactive liée au GDP. Des dérégulations de leur expression et de leur activation sont retrouvées dans de nombreux cancers. A ce jour, aucun inhibiteur sélectif de ces GTPases n'a pu être développé afin de bloquer l'activité de l'une ou l'autre de ces Rho. Ce travail doctoral a permis de mettre au point une approche innovante ciblant sélectivement l'état activé de la protéine RhoB. Suite à la caractérisation d'une nouvelle banque d'anticorps à simple domaine, sa validation par phage display contre divers antigènes a fourni de nombreux anticorps de haute fonctionnalité dans plusieurs applications. L'établissement d'une stratégie de sélection directe d'anticorps intracellulaire (intracorps) dirigés contre RhoB a permis d'identifier plusieurs intracorps conformationnels de la forme active de RhoB, dont un discriminant RhoB de ses homologues RhoA et RhoC. La fonctionnalisation d'intracorps par un domaine Fbox conduisant une dégradation de la cible a ensuite fourni la première stratégie efficace d'inhibition sélective de l'activité de RhoB. Ces travaux ont notamment démontré que l'extinction de l'activité de RhoB par intracorps fonctionnalisé augmente la migration et l'invasion de cellules pulmonaires. Ainsi cette avancée permettra de déterminer si l'activité de RhoB peut être une nouvelle cible thérapeutique et ouvre de nouvelles perspectives d'étude fine de l'activité des GTPases
RhoB GTPase shares more than 85% of homology with RhoA and RhoC. These proteins switch between an active conformation bound to GTP and an inactive one bound to GDP. Deregulations of their expression and/or their activity are often found in many cancers. To date, no selective inhibitor of these GTPases has been developed in order to block selectively Rho's activity. This project showed an original approach targeting RhoB's activity. After a new single domain antibody library characterization, its validation using the phage display technology against various antigens gave many highly functional antibodies in many applications. Set up of a new direct screening strategy of intracellular antibody (intrabody) raised against RhoB allowed us to identify several conformational intrabodies of RhoB active form, one of them discriminating RhoB from its homologs RhoA and RhoC. Intrabody functionalization with an Fbox domain driving target to degradation led to the identification of the first efficient selective RhoB activity inhibitory strategy. These work demonstrated that RhoB activity knockdown with functionalized intrabodies increased migration and invasion of pulmonary cells. In conclusion this tool will allow to determine if RhoB activity could be a new therapeutic target and open new perspectives to study GTPases activity

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8

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.

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9

Peurois, François. "Activation des petites GTPases à la périphérie des membranes". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLN037.

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Les petites GTPases sont des régulateurs majeurs de nombreux processus cellulaires. La dérégulation de l’activation des petites GTPases est à l’origine de nombreuses maladies comme, entre autres, certains diabètes et cancers. In vivo, l’activation des petites GTPases se fait par des facteurs d’échange nucléotidiques (GEF), qui interagissent avec les GTPases à la périphérie des membranes cellulaires. Au delà d’un simple lieu de co-localisation, les membranes biologiques possèdent des propriétés physico-chimiques impactant directement l’activation des petites GTPases par les GEFs. Ce projet de thèse s’articule autour de trois axes, 1) proposer une stratégie expérimentale pour mesurer quantitativement les effets des membranes dans cette activation, 2) établir un modèle d’activation à la périphérie des membranes du GEF EPAC1, cible thérapeutique de maladies cardiaques 3) caractériser des petites molécules inhibitrices connues d’ArfGEF dans un contexte membranaire. Les résultats ont montré que les membranes modifiaient l’efficacité catalytique des GEFs, et questionnait leur spécificité vis à vis des petites GTPases. Les membranes apparaissent également comme de véritables actrices de l’activation d’EPAC1 en coopération avec l’AMPc. Ces effets pourraient être expliqués par une colocalisation entre GEFs et GTPases à la surface des membranes, l’induction d’un réarrangement conformationnel du GEF par les membranes, une modification de la diffusion latérale des GEF, ou encore une géométrie catalytiquement avantageuse du complexe GEF-GTPase-membrane. Enfin comprendre et expliciter l’implication des membranes dans cette activation amène à imaginer de nouvelles stratégies d’inhibition thérapeutique
Small GTPases are major regulators of many cellular processes. Nucleotide exchange factors (GEF) activate small GTPases. Deregulation of the activation of small GTPases is at the origin of several diseases, such as certain diabetes and cancers. GTPases and GEFs interact together at the periphery of cell membranes. Beyond a simple place of co-localization, biological membranes have physicochemical properties directly impacting the activation of small GTPases by GEFs. This thesis project is based on three axes, 1) to propose an experimental strategy to quantitatively measure the effects of membranes in this activation 2) to establish a model of the activation at the periphery of membranes of the GEF EPAC1, a therapeutic target in heart diseases, 3) to characterize known ArfGEF inhibitory small molecules in a membrane context. The results showed that membranes modified GEF catalytic efficiency, and questioned their specificity towards small GTPases. The membranes also appear as partners for the activation of EPAC1 in cooperation with cAMP. These effects could be explained by a co-localization between GEF and GTPases on the membranes surfaces, a conformational rearrangement of the GEF induced by membranes, a modification of lateral diffusion of the GEF, or a catalytically advantageous geometry of the GEF-GTPase-membrane complex. Finally, understanding the involvement of membranes in this activation leads us to imagine new therapeutic inhibition strategies

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10

Keller, Laura. "Conception de nano-anticorps conformationnels comme nouveaux outils d'étude de l'activité des GTPases de la sous-famille RHOA". Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30005/document.

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Les GTPases de la sous famille RHOA participent à la régulation de nombreuses voies de signalisation qui contrôlent la dynamique du cytosquelette cellulaire et une grande diversité de fonctions telles que la prolifération, la division, la migration et la polarité cellulaires. Ce sont de véritables interrupteurs moléculaires qui, en réponse à un stimulus, changent de conformation tridimensionnelle pour activer leurs protéines effectrices cibles. Elles existent donc sous deux formes, une forme inactive liant le GDP et une forme active, liant le GTP. La proportion de forme active est extrêmement régulée au niveau spatial et temporel dans une cellule et représente moins de 10% de sa totalité. Depuis près de 20 ans, le seul outil disponible pour étudier leur activation est constitué par le domaine de liaison d'un effecteur, le RBD. Peu stable, faiblement soluble et peu adaptable, de nouveaux outils sont nécessaires afin de mieux comprendre la fine régulation de ces protéines. Les anticorps à simple domaine, VHH ou nanobodies, sont caractérisés par leur stabilité, solubilité, haut rendement de production et versatilité de fonctionnalisation. A partir d'une nouvelle banque d'anticorps à simple domaine optimisée pour la production d'intracorps, nous avons isolés différents clones capables de reconnaître in vitro et de bloquer in cellulo la forme active de ces protéines. L'un de ces clones permettra le développement d'un nouvel outil de mesure de l'activité de ces protéines in vitro tandis qu'un autre, in cellulo, permettra de mieux comprendre la régulation spatiale et temporelle des protéines endogènes
RHOA small GTPase belongs to a subfamily acting as a molecular switch activating major signaling pathways that regulate cytoskeletal dynamics and a variety of cellular responses such as cell cycle progression, cytokinesis, migration and polarity. RHOA activity resides in a few percent of GTP loaded protein, which is finely tuned by a crosstalk between regulators of the GTPase cycle. Manipulating a single RHO at the expression level often induces imbalance in the activity of other RHO GTPases, suggesting that more specific tools targeting these active pools are needed to decipher RHOA functions in time and space. We decided to use single domain antibodies, also known as VHH or nanobodies, as a new tool for studying RHOA activation. We produced and screened a novel fully synthetic phage display library of humanized nanobodies (NaLi-H1) to develop conformational sensors of the GTP loaded active conformation of RHO subfamily. We obtained several high affinity nanobodies against RHOA's active form which we characterized as RHO active antibodies in vitro and RHO signaling blocking intrabodies in cellulo. These new tools will facilitate and improve our current knowledge of this peculiar protein subfamily and will be a paradigm for the study of other RHO related small GTPases

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11

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.

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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 rythmique
Brugada 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

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12

Foucher, Anne-Emmanuelle. "Caractérisation biochimique de YphC, une protéine de Bacillus subtilis à deux domaines GTPases impliquée dans la biogenèse du ribosome". Grenoble, 2010. http://www.theses.fr/2010GRENV040.

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Les grands programmes de séquençage des génomes ont révélé l'existence de nombreux gènes de fonction inconnue. L'invalidation systématique de ces gènes chez les bactéries a permis de révéler le caractère essentiel de certains d'entre eux. L'étude des protéines issues de ces gènes s'est amplifiée ces dernières années car elles sont des cibles potentiellement intéressantes pour le développement de nouvelles molécules antibactériennes. YphC est une GTPase de Bacillus subtilis qui répond à ces critères. Elle est très conservée au sein des bactéries mais n'est pas retrouvée chez les organismes eucaryotes ou les archaebactéries, ce qui fait d'elle une cible de choix pour le développement de nouvelles molécules antibactériennes. YphC a la particularité de posséder deux domaines GTPases en tandem. Unique en son genre, nous avons voulu étudier cette protéine sous son aspect biochimique afin de mieux comprendre son mécanisme de fonctionnement. Nous avons donc mis au point la production et la purification de YphC et généré des mutations ponctuelles ou des délétions. Nous avons ainsi pu mesurer les constantes enzymatiques de cette protéine et caractériser l'effet d'activation du potassium sur son activité d'hydrolyse du GTP. Nous avons ainsi montré la forte activité GTPase de la protéine portée par le premier domaine GTPase et le rôle régulateur du deuxième domaine GTPase. Nous avons également étudié le rôle de YphC par une approche in vitro. Nous avons pu ainsi montrer que YphC est capable d'interagir avec les ribosomes de façon nucléotide dépendante suggérant un rôle de la protéine dans les processus de biogenèse du ribosome
Genome sequencing programs have revealed many genes of unknown function. The systematic disruption of these genes revealed the essentiality for some of them. Studying orphan proteins became of first importance as they are ideal targets for new antibacterial compounds. YphC is a GTPase from Bacillus subtilis that meets these criteria. It is well conserved throughout bacterial kingdom but is not found in eukaryota or archeas, strengthening the choice of this protein as a future target for antibacterial drugs. YphC has the particularity to possess two GTPase domains in tandem. As a unique protein, we decided to study YphC from a biochemical point of view to better understand its catalytic mechanism. We overexpressed and purified the protein, either wild type or mutants. We measured its enzymatic constants and characterize potassium activation effect on its hydrolytic activity. We showed that YphC displays a high GTPase activity and that GD1 bears the majority of this activity. GD2 would thus have a regulatory role in the protein. We also studied the role of YphC in vitro. We showed that the protein was able to interact with ribosome from Bacillus subtilis in a nucleotide dependant manner, suggesting that YphC plays a role in ribosome biogenesis

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13

Koraïchi, Faten. "Etude de l'activation de la GTPase RhoB par complémentation split-GFP tripartite". Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30081.

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RhoB est une petite GTPase rapidement activée par les facteurs de croissance et les stress cellulaires, qui régule des processus biologiques fondamentaux comme la migration, l'angiogenèse, la réparation de l'ADN, l'apoptose ainsi que la réponse à des thérapeutiques anticancéreuses. L'activité des petites GTPases est finement régulée par leur localisation subcellulaire. Cependant, l'activation de RhoB en cellules vivantes n'avait jamais été investiguée. Ce travail a permis d'adapter et de valider une méthode innovante d'analyse des interactions protéine-protéine par complémentation split-GFP tripartite, pour la détection sensible et spécifique de l'activation des petites GTPases en cellules vivantes. Nous avons ensuite développé un modèle cellulaire optimisé par la combinaison de la technologie split-GFP tripartite et d'un intracorps anti-GFP amplificateur de fluorescence, pour détecter la régulation de l'activation de RhoB avec une haute résolution spatiale. Ce biosenseur a mis en évidence la translocation de la forme active de RhoB en réponse au sérum à partir des endosomes pour s'accumuler au niveau de la membrane plasmique, révélant ainsi une nouvelle plateforme de signalisation membranaire de RhoB. Ce biosenseur permettra d'analyser le profil d'activation de RhoB et d'autres petites GTPases, sous d'autres stimulations ou dans différents contextes cellulaires, et d'identifier leurs partenaires et les modulateurs de leur activation
RhoB is a small GTPase that is rapidly activated in response to growth factors and cellular stress. It regulates fundamental biological processes such as cell migration, angiogenesis, DNA repair, apoptosis and response to anticancer therapies. Small GTPases activity is tightly regulated by their subcellular localization. However, RhoB activation had never been investigated in living cells. In this work, we have adapted and validated an innovative method of protein-protein interactions analysis using tripartite split-GFP complementation, for the sensitive and specific detection of small GTPases activation in living cells. Then, we developed an optimized cellular model by combining the tripartite split-GFP technology with an anti-GFP intrabody fluorescence-enhancer to detect the regulation of RhoB activation with high spatial resolution. This biosensor highlighted the translocation of active RhoB from endosomes to accumulate at the plasma membrane upon serum stimulation, revealing a novel membrane signaling platform of RhoB. Future studies based on this biosensor will enable the analysis of RhoB activation profile and other small GTPases upon various stimuli or in different cellular contexts, as well as the identification of the GTPases partners and activation modulators

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14

Vignal, Emmanuel. "Polymérisation de l'actine GTPases de la famille Rho : caractérisation et étude d'un effecteur de la Petite GTPase RhoG". Montpellier 2, 2001. http://www.theses.fr/2001MON20042.

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15

Mitteau, Romain. "Régulation par la phosphorylation d’un module Rho GTPase dans la levure Saccharomyces cerevisiae". Thesis, Bordeaux 2, 2013. http://www.theses.fr/2013BOR22084/document.

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Le cycle cellulaire eucaryote est caractérisé par des changements abrupts et dynamiques de la polarité cellulaire lorsque les chromosomes sont dupliqués et ségrégés. Ces évènements nécessitent une coordination entre la machinerie du cycle cellulaire et les régulateurs de la polarité. Les mécanismes qui contrôlent cette coordination ne sont pas totalement compris. Dans la levure S. cerevisiae, comme dans d’autres organismes eucaryotes, la GTPase Cdc42 joue un rôle important dans la régulation de la polarité cellulaire. En effet ses régulateurs constituent un module GTPase qui subit une phosphorylation dynamique, au cours du cycle cellulaire, par des kinases évolutivement conservées dont la Cycline-Dependent Kinase 1 (Cdk1) et la p21-Activated Kinase (PAK). Ces kinases et substrats pourraient relier la polarité et la progression dans le cycle cellulaire. En utilisant une approche in vitro, nous avons reconstitué la phospho-régulation du Guanine nucléotide Exchange Factor (GEF) de Cdc42, la protéine Cdc24. Nous avons identifié un possible mécanisme de régulation de la phosphorylation impliquant une protéine d’échafaudage qui augmente la phosphorylation de Cdc24 par la PAK et Cdk1. Cette phosphorylation accroit modérément l’affinité de Cdc24 pour cette même protéine d’échafaudage, Bem1. De plus, en testant les effets d’autres composants du module GTPase sur la phosphorylation de Cdc24, nous avons identifié un effet antagoniste pour une GTPase Activating Protein (GAP), Rga2. Cette protéine est présente dans le même complexe que Cdc24 et Bem1, les membres de ce complexe sont tous phosphorylés par Cdk1. Des mutants rga2 suggèrent que la phosphorylation que subie Rga2 inhibe son activité GAP. Nous proposons un modèle provisoire pour expliquer la présence de Rga2 dans ce complexe et l’inhibition qu’elle oppose à la phosphorylation de Cdc24. La présence de la protéine GAP dans le complexe pourrait être un mécanisme de contrôle de la phosphorylation de Cdc24 dans le but de déstabiliser son intéraction avec la protéine Bem1 en cas de mauvaise localisation du complexe. Par ailleurs, la PAK est activée par l’activité de Cdc42, nos résultats sont consistants avec un modèle dans lequel des signaux du cycle cellulaire engendreraient une auto-amplification de l’activation du module GTPase. Chez S. pombe, la croissance polarisée nécessite un gradient d’activation de Cdc42 dû à une ségrégation de GEF et de GAP. Dans ces travaux nous montrons que toutes les protéines GAPs de Cdc42 localisent aux sites de croissance au cours du cycle cellulaire. Ces localisations sont consistantes avec le besoin de cyclage de Cdc42 pour maintenir sa polarisation. Ces résultats suggèrent que la localisation des protéines GAP régulant Cdc42 chez S. cerevisiae semble différente de ce qui est connu chez S. pombe
The eukaryotic cell cycle is characterized by abrupt and dynamic changes in cellular polarity as chromosomes are duplicated and segregated. Those dramatic cellular events require coordination between the cell cycle machinery and polarity regulators. The mechanisms underlying this coordination are not well understood. In the yeast S. cerevisiae, as in other eukaryotes, the GTPase Cdc42 plays an important role in the regulation of cell polarity. Cdc42 regulators constitute a GTPase module that undergoes dynamic phosphorylation during the cell cycle by conserved kinases including Cyclin-Dependent Kinase 1 (Cdk1) and p21-activated kinase (PAK). These kinases and substrates may link cell polarity to the cell cycle progression. Using in vitro approaches, we have reconstituted the phospho-regulation of the Cdc42 Guanine Nucleotide Exchange Factor (GEF), Cdc24. We have identified a possible mechanism of Cdc24 regulation involving a scaffold-dependent increase in Cdc24 phosphorylation by Pak and Cdk1. This phosphorylation moderately increases the affinity of Cdc24 for another GTPase module component, the scaffold Bem1. Moreover, by testing the effect of other GTPase module components on the phosphorylation of Cdc24, and thus on its interaction with the scaffold, we identified an antagonistic function for the GTPase Activating Protein (GAP) Rga2. Our in vivo data of rga2 mutants suggest that Rga2 phosphorylation by Cdk1 inhibits its GAP activity. We propose a tentative model to explain the inhibition of Cla4 by Rga2 and its presence in a complex containing Cdc24 and Bem1. The presence of the GAP protein in the complex may be a mechanism that reduces Cdc24 phosphorylation in case of a mistargetting of the complex in order to downregulate the GEF/Scaffold dimer. Since the PAK component of the GTPase module is itself activated by Cdc42 activity, our results are consistent with a model in which inputs from the cell cycle lead to auto-amplification of the Cdc42 GTPase module. In S. pombe, polarised growth requires a gradient of activation of Cdc42 due to GEF and GAP segregation. Here we show that all Cdc42 GAPs localise to the polarised site during the cell cycle. Those localisations are consistent with a requirement of Cdc42 cycling to maintain a polarity cap. Our results may suggest that Cdc42 GAPs localisations in S. cerevisiae are different from current knowledge in S. pombe

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16

Tillement, Vanessa. "Régulation de la GTPase RHOB par phosphorylation". Toulouse 3, 2005. http://www.theses.fr/2005TOU30175.

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RhoB appartient à la famille Rho (RhoA, RhoB et RhoC) des GTPases de faible poids moléculaire, régulées par un cycle de liaison au GDP et GTP. Nous avons mis en évidence que RhoB est régulée également par phosphorylation. Contrairement à RhoA, qui est phosphorylée par la PKA, RhoB est, elle spécifiquement phosphorylée par la Caséine kinase 1 (CK1) et la Calmoduline kinase II in vitro et in vivo. Des études en spectrométrie de masse nous ont permis de montrer que CK1 phosphoryle RhoB dans son extrémité C-terminale sur la sérine 185. L'utilisation d'inhibiteurs de CK1 a permis de montrer que la phosphorylation de RhoB CK1-dépendante inhibe sa liaison à un des ses effecteurs, donc probablement son activité. Des résultats préliminaires suggèrent fortement que la phosphorylation de RhoB par CK1 est impliquée dans la régulation du trafic du récepteur à l'EGF suite à son internalisation par l'EGF
RhoB belongs to the Rho family (RhoA, RhoB and RhoC) of the low molecular weight GTPases, regulated by cycling between GDP and GTP bound state. We have shown that RhoB is also regulated by phosphorylation. On contrast to RhoA, which is phosphorylated by PKA, RhoB is specifically phosphorylated by Casein kinase 1 (CK1) and Calmodulin kinase II in vitro and in vivo. Mass spectrometry analysis has shown that CK1 phosphorylates RhoB on its C-terminal sequence on serine 185. With CK1 inhibitors we have shown that CK1-mediated phosphorylation of RhoB inhibits its binding to one of its effector, thus inhibiting its activity. Finally, preliminary results strongly suggest that RhoB phosphorylation by CK1 is implicated in the regulation of the intracellular trafficking of internalized EGF receptor

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17

Visvikis, Orane. "GTPase Rac1 et ubiquitination". Paris 5, 2007. http://www.theses.fr/2007PA05P622.

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Cette thèse a été consacrée à l’étude de la régulation par ubiquitination d’une protéine de signalisation cellulaire, la GTPase Rac1. J’ai montré que l’ubiquitination dégradative de Rac1 affecte peu son variant d’épissage Rac1b, et qu’elle requiert l'activité JNK, stimulée par Rac1 mais non par Rac1b. En parallèle, j’ai mis en évidence une ubiquitination non dégradative de Rac1 qui pourrait contribuer à l’internalisation bactérienne lors de l’invasion. En recherchant l’enzyme responsable de l’ubiquitination spécifique de Rac1, j’ai pu identifier la protéine à domaine RING finger Unkempt comme un nouvel effecteur de Rac1. Cette ubiquitine ligase potentielle, activée par Rac1, serait impliquée dans l’ubiquitination du facteur BAF60b appartenant au complexe chromatinien SWI/SNF. J’ai par ailleurs observé que Rac1 stimule la mono-ubiquitination de l’histone H2A. Ainsi, la GTPase Rac1 serait impliquée dans une ou plusieurs voie(s) de signalisation inédite(s) contrôlant le remodelage de la chromatine
This thesis has been dedicated to the study of the regulation by ubiquitination of a signaling protein, the Rac1 GTPase. I have shown that the degradative ubiquitination of Rac1 affects poorly its splice variant Rac1b, and requires JNK activity, which is stimulated by Rac1 but not by Rac1b. In addition, I have described a non-degradative ubiquitination of Rac1, which could participate in pathogen endocytosis during bacterial infection. Searching for the enzyme responsible for specific Rac1 ubiquitination, I have identified a RING finger protein, Unkempt, as a new effector of Rac1. I have shown that this potential ubiquitin ligase, which is activated by Rac1, could be involved in the ubiquitination of BAF60b, a component of the chromatin remodeling complex SWI/SNF. Moreover, I have observed that Rac1 stimulates histone H2A mono-ubiquitination. Thus, Rac1 GTPase could be involved in novel pathways by controlling chromatin remodeling

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18

Slaymi, Chaker. "Rôle de la GTPase atypique RhoU dans l'homéostasie intestinale". Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20127.

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L'épithélium intestinal se renouvelle tous les 4 à 6 jours chez les mammifères grâce aux cellules souches localisées au fond des cryptes. Le renouvellement dépend des signaux émis par le microenvironnement et requiert une phase de prolifération des cellules souches, de différenciation et d'apoptose/desquamation des cellules épithéliales. La signalisation Wnt, joue un rôle majeur dans l'homéostasie intestinale par l'action de deux gradients inversés le long de l'axe crypte/lumière ; la signalisation Wnt canonique, active au fond des cryptes, contrôle la prolifération alors que la signalisation non-canonique, active vers le haut des cryptes contrôle la différenciation. Il a été montré que ces deux voies contrôlent l'activité de la GTPase atypique RhoU/Wrch1. RhoU fait partie des GTPases qui s'activent spontanément, son activité est donc directement proportionnelle à son niveau d'expression dans la cellule. Enfin, cette GTPase atypique est sous exprimée dans de nombreuses tumeurs gastriques et colorectales.Compte tenu de ces données, nos objectifs étaient donc de caractériser les changements morphologiques induits par l'invalidation conditionnelle de RhoU dans l'épithélium intestinal murin et d'en déterminer les mécanismes d'action. Nos résultats montrent que la déplétion de RhoU n'est pas létale, cependant elle a induit une augmentation de 20% de la densité cellulaire et une désorganisation de la structure de l'épithélium dans le haut des cryptes du colon. Cette augmentation concerne aussi bien les lignages sécrétoires et absorptifs, cependant, l'absence de RhoU a induit une sur-représentation du lignage sécrétoire. Dans la lignée de tumeur colorectale DLD-1, nous avons montré que l'absence de RhoU mime le phénotype d'augmentation de la densité cellulaire observé chez la souris. L'invalidation de RhoU ne modifie pas la distribution des phases du cycle cellulaire ni de celle de la mitose, cependant, elle réduit le nombre des cellules en apoptose dans le colon des souris et dans les cellules DLD-1. L'invalidation de RhoU a réduit la signalisation Hippo et a altéré la contractilité cellulaire via une augmentation de la phosphorylation de la protéine MLC2. Des travaux récents ont montré que la diminution du niveau MLC2 phosphorylée est nécessaire pour l'activation des caspases par un stimulus apoptotique. Ceci suggère que cette perturbation de la contractilité peut être à l'origine de cette diminution de l'apoptose qui est la cause majeure responsable de ce phénotype. En conclusion, RhoU est un régulateur de l'homéostasie intestinale chez la souris via son rôle modérateur de la mort cellulaire
In Mammals, the intestinal epithelium is renewed every 4-6 days through the stem cells located at the bottom of crypts. The renewal depends on signals from the micro-environment and requires a proliferation phase of stem cells, then a differentiation and apoptosis/desquamation phases of epithelial cells. Wnt signaling plays a major role in intestinal homeostasis by the action of two reversed gradients along the axis crypt/ lumen: canonical Wnt signaling, active in the bottom of crypts, control proliferation while non canonical signaling, active in the top of the crypts control cell differentiation. It was shown that these two pathways are regulator of the atypical GTPase RhoU/Wrch1. The RhoU protein activates spontaneously, its activity is directly proportional to its expression level in the cell and is expressed as in gastric and colorectal tumors. In view of these informations, our objectives were therefore to characterizethe morphological changes induced by conditional invalidation of RhoU in the intestinal epithelium of mice and to determine the mechanisms of action. Our results show that RhoU depletion is not lethal. However, it induces an increase of cell density (+20%) and a disruption of the epithelium structure in the top of the colonic crypts. This increase affects both absorptive and secretory lineages. However, the absence of RhoU induced over-representation of secretory lineage. In colorectal tumor cell line DLD-1, we have shown that the absence of RhoU mimics the phenotype of cell density increase observed in mice. RhoU invalidationdid not change the distribution of cell cycle phases and mitosis, however, it reduces the number of apoptotic cells in the colon of mice and in the DLD-1 cells. RhoU invalidation reduced Hippo signaling and altered cell contractility via the increase of the protein MLC2 phosphorylation. Recent work has shown that the reduction of MLC2-P level is necessary for the caspase protein activation by an apoptotic stimulus. Suggesting that the perturbation of contractility may be the cause of this apoptosis decrease which is the main cause responsible of this phenotype. Finally, RhoU is a regulator of the intestinal homeostasis in micevia its moderating role of cell death

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Mercey, Olivier. "Interactions des microARN de la famille miR-34/449 avec les voies de signalisation intracellulaire : rôle dans la différenciation des cellules multiciliées chez les vertébrés". Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4119/document.

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Les cellules multiciliées (MCC) possèdent à leur surface apicale des centaines de cils mobiles générant un flux directionnel liquidien nécessaire par exemple pour le nettoyage des voies respiratoires. La fabrication de ces cils (multiciliogénèse) requiert une séquence d’évènements cellulaires dont un arrêt du cycle cellulaire, une réorganisation du réseau apical d’actine, une multiplication massive des centrioles suivie de leur migration au pôle apicale et de leur maturation en corps basal, à partir desquels les cils s’allongent.Mon laboratoire d’accueil a mis en évidence le rôle conservé de la famille de microARN miR-34/449 dans le contrôle de la multiciliogénèse en inhibant la voie de signalisation Notch ainsi qu’en induisant un arrêt du cycle. Au cours de ma thèse, j’ai mis en évidence un nouveau niveau de régulation de ces microARN par lequel ils contrôlent la réorganisation apicale du cytosquelette d’actine, en modulant l’expression et l’activité de certaines petites GTPases. Par ailleurs, j’ai identifié et caractérisé des séquences variantes des miR-34/449 canoniques, appelées isomiR. Tandis que ces isomiR partagent des fonctions semblables à celles de leurs homologues canoniques, ils apportent également une complémentarité d’action en modulant des transcrits cibles spécifiques. Enfin, le dernier axe de mon travail a permis d’identifier le rôle de la voie de signalisation BMP dans la multiciliogénèse ainsi que d’élucider certains des mécanismes moléculaires par lesquels elle contrôle ce phénomène. L’ensemble de nos découvertes offre une opportunité inédite pour développer des stratégies thérapeutiques dans le traitement de maladies associées à des désordres ciliaires
Vertebrate multiciliated cells (MCC) project hundreds of motile cilia at their apical surface which coordinately beat to generate a directional fluid flow necessary for many biological functions including airway cleansing. Biogenesis of multiple cilia (multiciliogenesis) follows different key cellular steps corresponding to a cell cycle arrest, a massive multiplication of centrioles which then migrate to the apical surface to form basal bodies, from which cilia elongate. In 2011, my host laboratory evidenced that the miR-34/449 family of microRNAs control vertebrate multiciliogenesis by inducing the cell cycle arrest and by repressing the Notch pathway. My thesis work has revealed a new role of miR-34/449 by demonstrating that they modulate expression and activity of small GTPases to drive the apical reorganization of the actin network, a prerequisite for basal body anchoring. Besides, I have identified and characterized variant sequences of canonical miR-34/449 family, named isomiRs. Whereas these isomiRs share common biological functions with canonical miR-34/449 miRNAs, they may also contribute to a complementary effect by targeting specific transcripts. Finally, the last part of my work has contributed to the identification of the conserved role of the BMP pathway in the control of multiciliogenesis. I have evidenced some molecular mechanisms by which the BMP signal controls this phenomenon. Importantly, I demonstrated that BMP inhibition promotes regeneration of tracheal MCC in vivo in an asthmatic mouse model. Overall, our findings offer an unprecedented opportunity to develop novel therapeutic strategies to treat diseases associated with ciliary disorders

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20

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.

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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.

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21

Paysan, Lisa. "Implication de la protéine Rnd3/RhoE dans la physiologie et la carcinogenèse hépatiques". Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0275/document.

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L'étude des mécanismes moléculaires de la carcinogenèse hépatique a montré l'implication de la RhoGTPase, Rnd3/RhoE. La protéine Rnd3 est sous-exprimée dans le carcinome hépatocellulaire et la diminution de son expression engendre, in vitro, une augmentation de l'invasion des hépatocytes tumoraux. Sur la base de ces travaux, ce projet de thèse s'est décomposé en deux axes. Le premier axe a été d'étudier le rôle de Rnd3 dans la carcinogenèse ainsi que dans la physiologie hépatique in vivo. Ce projet a débuté par la génération d'un modèle murin présentant un KO conditionnel ethépato-spécifique de Rnd3 {KORnd3). L'utilisation de plusieurs stratégies s'est révélée nécessairepour obtenir une extinction protéique de Rnd3 dans la majorité des hépatocytes chez les souris KO. Après hépatectomie des deux tiers, les premiers résultats montrent un retard de régénération hépatique chez les souris KORnd3. En ce qui concerne la carcinogenèse hépatique, nous avons mis en place un modèle de carcinogène chimique en utilisant le diéthylnitrosamine et un modèle de carcinogénèse spontanée chez les animaux KORnd3Hep. Le deuxième axe a porté sur l'étude des invadosomes, structures d'actine impliquées dans l'invasion cellulaire. Nous avons établi une signature minimum pour les invadosomes, impliquant la GTPase Cdc42 et la protéine d'échafaudage TksS. Nos résultats suggèrent également une implication de Rnd3 dans la fonction de dégradation des invadosomes. Ce travail de thèse a ainsi permis d'apporter de nouveaux outils et de nouvellespistes quant à l'implication de Rnd3 dans la physiopathologie hépatique et dans l'invasion cellulaire
The study of the molecular mechanisms involved in hepatic carcinogenesis revealed the significant down-regulation of the RhoGTPase Rnd3/RhoE in hepatocellular carcinoma as compared to non- tumor liver. Rnd3 down-regulation provides an invasive advantage to tumor hepatocytes suggesting that RND3 might represent a metastasis suppressor gene in hepatocellular carcinoma. This PhD work was divided in two axes. We first studied the role of Rnd3 in the mouse liver using carcinogenesis and liver regeneration protocols. We thus generated conditional and liver specific Rnd3 KO mice (KORnd3Hep). The first results obtained after partial hepatectomy suggest a delay in liver regeneration for the KORnd3Hep mice. We also developed a carcinogenesis strategy in KORnd3Hep mice using diethylnitrosamine treatment. The second axis focused on invadosomes, which are actin-based structures involved in cell invasion. We have determined a minimal and universal molecular signaturefor functional invadosomes, which involves the RhoGTPase Cdc42 and the adaptor protein TksS. We also highlighted the role of Rnd3 in invadosome degradation. ln conclusion, this work provides new tools and new insights on Rnd3 function in hepatic physiopathology and cellular invasion

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22

Seviour, Elena Genevieve. "Regulation of the small GTPASE ARF6". Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492445.

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ADP ribosylation factors (ARFs) are a family of small GTPases from the ras superfamily. 6 members of the ARF family have been identified in mammals to date, with functions ranging from the regulation of vesicular coat protein assembly to the organisation of the actin cytoskeleton. ARFs have no intrinsic GDP/GTP exchange or GTPase activity, and so their GDP/GTP cycle is dependent on regulation by exchange factors and activating proteins.

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23

Atkinson, Gemma C. "Evolution of the translational GTPase superfamily". Thesis, University of York, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479486.

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Davidson, Anthony Christopher. "Salmonella manipulation of Arf GTPase networks". Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708908.

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25

Wu, Guang. "ROP GTPASE signaling in tip growth /". The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486546889380932.

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26

Zhang, Yong. "Spatial regulation of motility in the social bacterium Myxococcus xanthus". Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22110.

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Tous les organismes, les animaux, les plantes et les microbes, sont composés de cellules polarisées, en affichant un positionnement asymétrique des organites sub-cellulaires ou des structures. Le contrôle de polarité a été étudié chez les eucaryotes pendant une longue période, et a été montré pour être impliqués dans de nombreux processus physiologiques, tels que l'embryogenèse, le cancer métastatique et les maladies dégénératives des neurones. Chez les procaryotes, des études de polarité ne sont apparues récemment avec le développement de la microscopie à fluorescence sensibles. Ces études ont révélé que les cellules procaryotes sont en fait très organisé et une masse croissante de la littérature a montré que les cellules bactériennes également utiliser des radeaux lipidiques, courbure membranaire, la paroi cellulaire et un cytosquelette complexe pour diriger le positionnement spécifique de structures subcellulaires.Petites GTPases de la superfamille Ras sont des éléments réglementaires polarisation répandue chez les eucaryotes. Malgré l'existence depuis longtemps de ces petites GTPases dans les génomes procaryotes, leur fonction a jamais été étudiée. Pendant ce travail de thèse, nous avons trouvé, pour la première fois, qu'une petite GTPase, MglA et de sa protéine apparentée Activation GTPase (GAP) MglB, directe une dynamique axe antéro-postérieur à la motilité directe en forme de tige deltaproteobacterium Myxococcus xanthus. Dans ce processus, MglA s'accumule dans son état lié au GTP au niveau du pôle leader de cellules, en activant les machineries motilité. Ce schéma de localisation est maintenue par MglB, qui localise le pôle opposé, le blocage de l'accumulation MglA à ce pôle à travers son activité GAP. Remarquablement, les deux protéines passer leur localisation synchrone, ce qui correspond à un changement dramatique dans la direction du mouvement cellulaire (inversion). Ce commutateur est réglementé par un système chimiosensoriels-like, Frz. Dans une deuxième partie de ce travail, nous avons identifié un régulateur de protéine de réponse, RomR qui est essentiel pour le regroupement polaire de MglA. Interdépendances complexes entre la localisation RomR, MglA et MglB indiquent que ces protéines pourraient constituer un complexe de polarité dynamique de trois protéines qui reçoit Frz de signalisation pour passer l'axe de polarité. En conclusion, les résultats de ce travail de thèse suggère que M. xanthus intégré un module de polarité eucaryotes-like (MglAB) dans un procaryote spécifique (Frz) réseau de signalisation pour réguler sa motilité. Une telle réglementation est distincte sous forme de petites protéines G des règlements, qui sont généralement couplés à la protéine G récepteurs couplés (GPCR) chez les eucaryotes. Enfin, ce travail ouvre la voie pour comprendre comment la réglementation seule la motilité cellulaire sont intégrés pour générer des comportements commandés multicellulaires donnant naissance à des structures primitives de développement, par exemple, la morphogenèse du corps fructifères. D'autre part, ce travail fournit également un exemple d'analyser les étapes évolutives donnant lieu à des réseaux de signalisation
All organisms, animals, plants and microbes, are composed of polarized cells, displaying asymmetric positioning of sub-cellular organelles or structures. Polarity control has been studied in eukaryotes for a long time, and has been shown to be involved in many physiological processes, such as embryogenesis, cancer metastasis and neuron degenerative diseases. In prokaryotes, polarity studies only emerged recently with the development of sensitive fluorescent microscopy. These studies revealed that prokaryotic cells are in fact highly organized and a growing body of literature has shown that bacterial cells also use lipid rafts, membrane curvature, the cell wall and a complex cytoskeleton to direct the specific positioning of subcellular structures.Small GTPases of the Ras superfamily are widespread polarization regulatory elements in eukaryotes. Despite the long known existence of such small GTPases in prokaryotic genomes, their function has never been studied. During this thesis work, we found, for the first time, that a small GTPase, MglA and its cognate GTPase Activating Protein (GAP) MglB, direct a dynamic anterior- posterior axis to direct motility of the rod-shaped deltaproteobacterium Myxococcus xanthus. In this process, MglA accumulates in its GTP-bound state at the leading cell pole, activating the motility machineries. This localization pattern is maintained by MglB, which localizes at the opposite pole, blocking MglA accumulation at this pole through its GAP activity. Remarkably, both proteins switch their localization synchronously, which correlates with a dramatic change in the direction of cell movement (reversal). This switch is regulated by a chemosensory-like system, Frz. In a second part of this work, we identified a response regulator protein, RomR which is essential for the polar clustering of MglA. Intricate localization interdependencies between Romr, MglA and MglB indicate that these proteins might constitute a dynamic three-protein polarity complex that receives Frz-signaling to switch the polarity axis. In conclusion, the results from this thesis work suggest that M. xanthus integrated a eukaryotic-like polarity module (MglAB) into a prokaryotic- specific (Frz) signaling network to regulate its motility. Such regulation is distinct form small G- protein regulations, which are generally coupled to G-protein coupled receptors (GPCRs) in eukaryotes. Finally, this work paves the way to understand how single cell motility regulations are integrated to generate ordered multicellular behaviors giving rise to primitive developmental structures, for example fruiting body morphogenesis. On the other hand, this work also provides an example to analyze the evolutionary steps giving rise to signaling networks

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27

Cabrejos, Diego Antonio Leonardo. "Especificidade na montagem de filamentos de Septinas: o caso da interface G entre SEPT5 e SEPT8". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-27102016-102703/.

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Septinas abrangem uma família conservada de proteínas que ligam e hidrolisam GTP e formam heterofilamentos, anéis e redes para realizar as suas funções. Apresentam três domínios estruturais: o domínio N-terminal contendo uma sequência polibásica (para ligar membranas), o domínio de ligação ao nucleotídeo (G) e o domínio C-terminal que inclui uma sequência predita de formar um coiled-coil. Em humanos, as 13 septinas são classificadas em quatro grupos (I, II, III e IV) baseadas nas sequências de aminoácidos. O único filamento caracterizado estruturalmente, até hoje, é o formado por SEPT2-SEPT6-SEPT7, mostrando que as subunidades interagem através de duas interfaces (chamadas G e NC). Os determinantes estruturais da montagem correta do filamento são pouco conhecidos, sendo o estudo limitado pela complexidade em purificar e cristalizar complexos triméricos ou tetraméricos. Uma abordagem alternativa é estudar interfaces individuais de um filamento (G e/ou NC) por separado. Assim, o presente projeto objetivou estudar, utilizando uma abordagem biofísica e estrutural, a interface G formada por SEPT5 e SEPT8 para elucidar os fatores importantes em determinar a sua especificidade. Os domínios GTPase de SEPT5 e SEPT8 foram clonadas em vetor de expressão bicistrônico pET-Duet, co-expressas e co-purificadas. Estudos de análise do estado oligomérico e homogeneidade foram conduzidos utilizando cromatografia de exclusão molecular, espalhamento dinâmico de luz e ultracentrifugação analítica, revelando um complexo dimérico e monodisperso. O complexo apresenta uma mistura aproximadamente equimolar de nucleotídeos (GTP e GDP) ligados enquanto SEPT8(G) sozinha é incapaz de ligar qualquer um dos dois. Além disto o complexo apresenta uma termoestabilidade maior que SEPT8(G), verificado por um aumento em Tm de 5°C. Com o intuito de observar os determinantes estruturais da especificidade, ensaios de cristalização foram conduzidos e assim, cristais do complexo SEPT5-SEPT8(G) que difrataram apenas a muito baixa resolução foram obtidos. Na ausência de uma estrutura cristalográfica, modelagem por homologia foi realizada para analisar as interfaces G entre diferentes combinações de septinas. Identificamos uma interação entre aminoácidos característicos (aminoácidos únicos para cada grupo de septinas) para o complexo formado entre membros do grupo III, (incluindo SEPT5) e membros do grupo II, (incluindo SEPT8). Esta interação entre Phe131 (grupo III) e Thr19 (grupo II) pode explicar a especificidade na formação de uma interface G entre septinas destes grupos durante a formação do filamento e além disso, a importância da presença do GTP ligado ao septina do grupo II. Com isto, propomos pela primeira vez uma explicação plausível da relevância da perda de atividade catalítica das septinas deste grupo, um fato inexplicado até o momento. Mutação dos resíduos identificados levou a uma mudança no seu perfil de eluição do complexo durante purificação por exclusão molecular indicando alterações na formação do complexo mutante.
Septins are a conserved family of proteins that bind and hydrolyze GTP and form heterofilaments, rings and networks in order to carry out their functions. They have three structural domains: an N-terminal domain containing a polybasic sequence (for membrane binding), a nucleotide-binding (G) domain and a C-terminal domain including a sequence predicted to form a coiled-coil. In humans, 13 septins have been classified into four groups (I, II, III and IV) based on their amino acid sequences. The only structurally characterized filament described to date is formed by SEPT2-SEPT6-SEPT7, which reveals that the subunits interact through two different interfaces (G and NC). The structural determinants of correct filament assembly are poorly known, and this is limited by the complexity of purifying and crystallizing trimeric or tetrameric complexes. An alternative approach is to study a single filament interface (G or NC) on its own. Here, we aimed to study, using biophysical and structural approaches, the G interface formed between SEPT5 and SEPT8 to elucidate the factors relevant to determining its specificity. The GTPase domain of SEPT5 and SEPT8, were cloned into the bicistronic expression vector pET-Duet, co-expressed and co-purified. Studies to determine the oligomeric state and homogeneity of the complex were conducted using size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation, revealing a monodisperse dimer for SEPT5-SEPT8(G). The complex elutes with an approximately equimolar mixture of bound nucleotides (GTP and GDP) whereas SEPT8(G) alone is shown to be unable to bind either. Furthermore, the complex has a greater thermostability than SEPT8(G), demonstrated by an increase of 5°C in Tm. In order to determine the structural determinants of specificity, crystallization trials were conducted and crystals of the SEPT5-SEPT8(G) complex were obtained, but these diffracted to only very low resolution. In the absence of a crystal structure, homology modeling was performed to analyze the potential G interfaces between different septin combinations. An interaction between characteristic amino acids (those which are unique to given septin group) was identified for the complex formed between group III septins (including SEPT5) and group II septins (including SEPT8). This interaction, between Phe131 (group II) and Thr19 (group III) may explain the specificity in the formation of a G interface between septins of these groups during filament formation and furthermore the importance of GTP bound to the group II septin. These observations allow us to propose for the first time a plausible explanation for relevance of the loss of catalytic activity by this septin group, an unexplained fact up until now. Mutation of the identified residues resulted in a change in the elution profile of the complex from the size exclusion column suggesting structural alterations in the mutants.

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28

Singh, Komudi. "Oxidant-Induced Cell Death Mediated By A Rho Gtpase In Saccharomyces cerevisiae". Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view.cgi?acc%5Fnum=osu1227716169.

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29

Aresta, Sandra. "Etude de la protéine Gem, un membre de la superfamille Ras". Paris 11, 2001. http://www.theses.fr/2001PA112281.

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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 Rho
Gem 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

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30

Kawano, Yoji, Takeshi Yoshimura y Kozo Kaibuchi. "Smooth muscle contraction by small GTPase Rho". Nagoya University School of Medicine, 2002. http://hdl.handle.net/2237/5374.

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Nozawa, Atsuko. "Rab35 GTPase recruits NPD52 to autophagy targets". Kyoto University, 2018. http://hdl.handle.net/2433/230994.

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32

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.

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Calcium (Ca Â²?) is a highly versatile signal that regulates a host of intracellular events across the biological spectrum. Carefully regulated changes in intracellular CaÂ²? 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.

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33

Kirsten, Marie Lis. "Biophysical studies of Rab GTPase membrane binding". Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6964.

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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.

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34

Silva, Patricia Maria de Oliveira e. "Contrôle spatio-temporel de la croissance filamenteuse chez Candida albicans". Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4030.

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Candida albicans est un pathogène fongique opportuniste de l’Homme, qui peut causer des infections superficielles mais aussi systémiques chez les patients immunodéprimés. Sa virulence est associée à sa capacité de changer d’une forme bourgeonnante à une forme hyphale. La petite GTPase de type Rho, Cdc42, est critique pour la croissance filamenteuse et, sous forme activée, sa localisation est restreinte à l’extrémité des hyphes. J’ai utilisé un système photoactivable, constitué des domaines d’Arabidopsis thaliana Cry2PHR-CibN, pour contrôler le recrutement de Cdc42 constitutivement actif à la membrane plasmique. J'ai déterminé comment le photo-recrutement de Cdc42 constitutivement actif perturbe la croissance filamenteuse et où, quand et comment une nouvelle croissance filamenteuse est ré-initiée. Mes résultats démontrent que, lors du photo-recrutement de Cdc42 constitutivement actif, l'extension du filament cesse puis un nouveau site de croissance s’établit dans la cellule. La localisation de ce nouveau site de croissance est corrélée à la longueur du filament. J'ai étudié les mécanismes moléculaires qui sous-tendent le désassemblage du site de croissance initial et l'emplacement spécifique du nouveau site de croissance filamenteuse. Dans les hyphes en croissance, un «cluster» de vésicules, appelé Spitzenkörper, est localisé à l'extrémité du filament. Lors du photo-recrutement de Cdc42 constitutivement actif, un nouveau «cluster» de vésicules, de composition similaire à celui du Spitzenkörper initial, apparaît dans la cellule mère. J'ai suivi la dynamique du Spitzenkörper et la localisation de Cdc42 sous forme activée, des sites d'endocytose, des vésicules de sécrétion et des câbles d’actine suite à la perturbation du site de croissance initial dans le filament. Dans l’ensemble, mes résultats indiquent qu'il existe une compétition pour la croissance entre le Spitzenkörper et le «cluster» de vésicules qui se forme immédiatement après le photo-recrutement de Cdc42 constitutivement actif et qu'un axe de polarité dynamique peut être établi en l'absence de croissance directionnelle
Candida albicans is a fungal human pathogen that can cause life-threatening infections in immunocompromised patients, in part, due to its ability to switch between an oval budding form and a filamentous hyphal form. The small-Rho GTPase Cdc42 is crucial for filamentous growth and, in its active form, localizes as a tight cluster at the tips of growing hyphae. I have used a light-activated membrane recruitment system comprised of the Arabidopsis thaliana Cry2PHR-CibN domains to control the recruitment of constitutively active Cdc42 to the plasma membrane. I have determined how photorecruitment of constitutively active Cdc42 perturbs filamentous growth and where, when and how new filamentous growth is subsequently initiated. My results demonstrate that, upon photorecruitment of constitutively active Cdc42, filament extension is abrogated and a new growth site can be established in the cell. Location of a new filamentous growth site correlates with the length of the initial filament. I have investigated the molecular mechanisms that underlie the disassembly of an initial growth site and the specific location of the new filamentous growth site. In growing hyphae a cluster of vesicles, referred to as a Spitzenkörper, is localized at the tip of the filament. Upon photorecruitment of constitutively active Cdc42, a new cluster of vesicles, with a composition similar to that of the initial Spitzenkörper, appears in the mother cell. I have followed the dynamics of the Spitzenkörper, active Cdc42, sites of endocytosis, secretory vesicles and actin cables subsequent to disruption of the initial growth site in the filament. Taken together, my results suggest that there is competition for growth between the Spitzenkörper and the cluster of vesicles that forms immediately after the photorecruitment of constitutively active Cdc42 and that a dynamic polarity axis can be established in the absence of directional growth

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35

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.

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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 hyphale
Candida 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

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36

Guillot, Charlene. "Etude du maintien de l'adhérence dans les tissus prolifératifs". Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4029.

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Les tissus épithéliaux présentent deux caractéristiques majeures, ils sont robustes (rôle de barrière) mais également plastiques lors de la morphogénèse. L'homéostasie des tissus épithéliaux repose sur la régulation de la balance prolifération/mort cellulaire. Dans ma thèse, je décris tout d'abord, les mécanismes moléculaires permettant à la cellule épithéliale de se diviser tout en maintenant l'intégrité du tissu. J'ai ensuite altéré cette intégrité, en utilisant le système de génération de clônes mosaïques, afin de comprendre comment la cohésion du tissu est maintenue. Ce travail m'a alors permis de comprendre comment l'adhérence est modulée, puis restaurée, au cours de la division cellulaire. Ainsi, j'ai montré que l'intégrité des tissus est assurée par l'action concomitante des forces d'adhésion et des forces de tension. Enfin, mon travail apporte également des éléments clés pour l'étude de la perte d'adhérence des cellules tumorales responsable en partie, de la progression des tumeurs solides en métastases
Tissue homeostasis relies on the tight regulation of cell proliferation and cell death. Epithelial tissues are robust tissues that support the structure of developing embryos and adult organs and are effective barriers that physically protect the organism against pathogens. In my thesis, I have first described the molecular mechanisms responsible for maintaining tissue integrity during epithelial cell division. I have then abrogated this integrity by inducing mosaic clones within tissues to understand how tissue cohesion is maintained. This work shows how the continuity of adhesive properties is ensured during cell division. It also reveals new key elements that result in loss of adhesion in tissues and thus may be responsible for the progession from solid cancer to metastasis

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37

Gloor, Yvonne. "The Arf GTPase exchange factor Sec7p interaction network:". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1202827858817-78960.

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The Golgi apparatus is the main crossroad of the intracellular trafficking network in all eukaryotic cells and plays a crucial role in the distribution of cellular material. To ensure the proper sorting and delivery of cargo proteins to their destination while maintaining Golgi homeostasis the coordination of all transport events to and from this organelle is required. Although a cascade of activation events has already been reported for Golgi Ypt/Rab proteins that function in the exocytic pathway, their connection to incoming vesicles from endosomal compartments or to the different Arf mediated vesicle formation machineries has still to be established. In addition, the role of lipids and the interplay between lipid and protein regulators at the Golgi are largely missing. In the present study, we used several approaches to unravel the crosstalk between known regulators of Golgi trafficking and to identify new proteins involved in this process. As starting point, we considered the results from four different screens before focusing on the role of Arf exchange factors. We report two new physical interactors of the late Golgi Arf-GEF Sec7p: the lipid kinase Pik1p and the cyclic nucleotide phosphodiesterase Cpd1p. In addition, our studies on the function of Sec7p revealed additional feature of this protein and it’s relationship to the other yeast Golgi Arf-GEFs. Arf proteins and their regulators play an important role in the formation of vesicles at the exit from the Golgi apparatus. There are three Golgi-localized Arf-GEFs in S.cerevisiae, Sec7p and the redundant Gea1p/Gea2p. While it has been established that Sec7p function does not overlap with the Gea’s, the specific role of these proteins remains unclear. We show that Sec7p colocalizes poorly with the Gea’s, indicating that these proteins activate Arf on different Golgi sub-compartments. In addition, our data suggest that Sec7p mainly promotes the formation of post-Golgi transport vesicles supporting forward transport from the late Golgi while the Gea’s primarily regulate COPI-mediated retrograde traffic. This observation is consistent with published data from mammalian cells and suggests that the spatial and temporal regulation of Arf is conserved from yeast to mammals. Both Arf regulation and phosphatidylinositol 4-phosphate (PI4P) metabolism are important factors for Golgi function. Here, we show that the yeast PI4-kinase, Pik1p binds specifically to Sec7p but not Gea1p or Gea2p. Taken together, the physical interaction, the colocalization and similar transport phenotypes of the respective mutants suggests a functional link between Pik1p and Sec7p but not the Gea’s. In addition, Pik1p binds to the catalytic domain of Sec7p and could directly influence the activity of the GEF. We propose that this interaction coordinates Arf activation with PI4P production to generate a highly specific dual recognition system for the recruitment of specific effectors to the late Golgi. Besides its catalytic domain, Sec7p shares several conserved regions with other members of the BIG/GBF Arf-GEF subfamilies, including the N-terminal DCB (Dimerization/Cyclophilin Binding) domain. We show that a single point mutation in the DCB domain of Sec7p efficiently inhibits Arf activation without affecting membrane recruitment of the GEF and could interfere with a possible dimerization of the protein. We identified Cpd1p as an allele specific dosage suppressor of the Sec7p DCB domain mutation. Cpd1p and Sec7p physically interact and both proteins localize independently to the late Golgi. Increased Golgi level of Cpd1p compensates for the loss of interaction due to the mutation in the DCB domain of Sec7p. The catalytic activity of Cpd1p is important for the rescue, indicating an intriguing connection between the Arf activation cycle and ADP-ribose derivates. We also find that Cpd1p interacts with several other proteins involved in Golgi- and post-Golgi transport events. Hence, Cpd1p is a new regulator of vesicular traffic at the Golgi that could act as a scaffolding factor for Sec7p and other transport proteins.

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38

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.

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39

Saunders, Amy Elizabeth. "GIMAP1 : a small GTPase involved in lymphocyte survival". Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612535.

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40

Erasmus, Jennifer Carr. "Small Pho GTPase Signalling Downstream of E-cadherin". Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503823.

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41

Graham, Deborah Louise. "The catalytic mechanism of Rho-GTPase-activating protein". Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322212.

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42

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.

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43

Kwiatkowska, Aneta, Sebastien Didier, Shannon Fortin, Yayu Chuang, Timothy White, Michael Berens, Elisabeth Rushing et al. "The small GTPase RhoG mediates glioblastoma cell invasion". BioMed Central, 2012. http://hdl.handle.net/10150/610207.

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BACKGROUND:The invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.RESULTS:We found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.CONCLUSIONS:Our functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.

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44

Bousquet, Emilie. "Rôle de la GTPase RhoB dans l'oncogenèse pulmonaire". Toulouse 3, 2010. http://thesesups.ups-tlse.fr/1007/.

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Le cancer bronchique est la première cause de mortalité par cancer dans le monde et ceci est largement dû à des diagnostics tardifs et à un fort pouvoir métastatique. L'amélioration de la prise en charge de ce type de cancer implique donc une meilleure connaissance des processus d'oncogenèse et d'invasion tumorale. Il a été préalablement montré au laboratoire que la surexpression de la GTPase RhoB inhibe la transformation cellulaire dans des cellules exprimant Ras et une correlation inverse entre l'expression de RhoB et la progression du cancer bronchique, cette perte d'expression se produisant entre les stades de carcinomes in situ et de carcinomes invasifs. Afin de définir plus précisément le rôle de RhoB dans les différentes phases de l'oncogénèse bronchique, l'objectif de mes travaux de thèse a été d'analyser l'effet de son inhibition sur le phénotype de cellules pulmonaires permettant ainsi de mimer la perte d'expression de la protéine. Ces travaux ont permis de montrer que l'inhibition de RhoB n'induit pas la transformation des cellules bronchiques mais stimule le potentiel migratoire et invasif des cellules bronchiques ainsi que le mode de migration. Par ailleurs, ces travaux ont permis de mettre en évidence que RhoB module le phenotype migratoire et invasif via la régulation en cascade de la kinase Akt1 et de la GTPase Rac1. L'ensemble de ces travaux suggèrent que RhoB est impliquée dans la progression tumorale, notamment sur l'acquisition des propriétés migratoires et invasives, plutôt que dans l'initiation de l'oncogenèse pulmonaire et que RhoB a un rôle critique dans la dissémination métastatique du cancer bronchique
Lung cancer is the leading cause of cancer-related death worldwide and this is largely due to late diagnosis and a high metastatic potential. This highlights the need for a better understanding of the molecular mechanisms involved in lung cancer initiation and progression. It has previously been shown in the laboratory that overexpression of RhoB GTPase inhibits cell transformation in cells expressing Ras and an inverse correlation between RhoB expression and lung cancer progression, this loss of expression occurs between carcinoma in situ and invasive carcinoma. To define more precisely the role of RhoB in different stages of lung oncogenesis, we have analysed the effect of its inhibition on the phenotype of lung cells to mimic the loss of RhoB. This work showed that inhibition of RhoB does not induce transformation of bronchial cells but increases migratory and invasive capabilities of bronchial cells and the migratory phenotype. Moreover, this work has helped to show that RhoB regulates the migratory and invasive phenotype via the regulation of Akt1 and Rac1 GTPase. The RhoB inhibition in bronchial cells, induced by the Ras oncogene, frequently mutated in Iung cancer, critical for stimulation of their migratory and invasive properties through the regulation of the kinase Akt1. All these data suggest that RhoB is involved in tumor progression, notably the acquisition of migratory and invasive properties, rather than in the initiation of pulmonary carcinogenesis and that RhoB has a critical role in the metastasis of lung cancer

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45

Hu, Shuang. "Rho GTPase Signaling Modulates Neurotransmission in Caenorhabditis elegans". University of Toledo / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1365182177.

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46

Li, Hai. "ROP GTPASE signaling in cell development of Arabidopsis /". The Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488191667181129.

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47

Patwardhan, Anand. "Role of the small GTPase RAB6 in pigmentation". Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCB090.

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48

Kamkar, Fatemeh. "Pftaire1 (Cyclin Dependent Kinase14): Role and Function in Axonal Outgrowth During the development of the CNS". Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32860.

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Cyclin Dependent Kinase (Cdk) family members play a role in CNS development. Cyclin Dependent Kinase 5 (Cdk5) is well known for its fundamental role in neuronal development and axogenesis, as well as, cell death. Other Cdks include Pctaire and Pftaire. Inhibition of Pctaire results in increased axon outgrowth, however, the role and function of Pftaire is unknown. Pftaire1 is a novel member of the Cdk family that was initially detected in a screen for cdc2-like kinases. Unpublished data from our lab reveals that Pftaire1 (Eip63E) deficiency in Drosophila melanogaster results in defects in the axon and neuronal structure of the ventral nerve cord (VNC). In mammals, Pftaire1 is highly, expressed in the CNS. Here, we proposed that Pftaire1 might have a role in axon outgrowth. To investigate the role of Pftaire1 in mammals, the first germline Pftaire1 knockout mice were generated. Considering the severe effects of Eip63E deficiency in Drosophila and the homology between mammalian and fly Pftaire1, CNS defects in the mouse were anticipated. However, to date, no gross abnormalities have been detected in the overall morphology, fertility, life span, or anatomical brain structures of the Pftaire1 deficient mice. This may be due to the presence of other post-mitotic Cdk proteins that are highly similar to Pftaire1. For instance, mammals possess Pftaire (1, and 2), as well as, Pctaire (1, 2, and 3), while Drosophila only possess the Pftaire1 orthologue where the Pftaire2 and Pctaire (1, 2, and 3) are absent. Furthermore, the mice were of mixed background. In spite of this, we demonstrated that Pftaire1 deficient neurons showed increased axon length, in the initial phases of culture. This was confirmed by expression of dominant negative (DN) D228N-Pftaire1 in wild type neurons. Also classification of axons into different ranges, reveals a higher percentage of hyperextended neurites in D228N and Pftaire1 knockout mice. The mechanism by which Pftaire1 controls axon outgrowth is unknown. In this study we show that, Pftaire1 interacts physically with the small GTPase proteins Rac1, Cdc42, and RhoA. Importantly, we showed that Pftaire1 phosphorylates GDP-RhoA on a serine residue. We propose that this regulates RhoA activity, which in turn controls axon outgrowth.

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49

Sørmo, Christopher. "Rho GTPases in Plants: Structural analysis of ROP GTPases; genetic and functionalstudies of MIRO GTPases in Arabidopsis thaliana". Doctoral thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for biologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-12507.

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50

Verma, Sunil Kumar. "Studies on the signalling of the small GTPase Rap1". Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491743.

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RaplA (Krev-l) is a member of the Ras superfamily of small GTP-binding proteins and has highest homology to Ras. Rapl behaves as an antagonist of oncogenic K-Ras activity in NIH3T3 cells and of polyoma middle-T antigen in Rat-2 cells. It inhibits transformation of human prostate cancer cells, hamster pancreatic adenocarcinoma cells and of ethylcarbamate induced lung adenomas in transgenic mice. However, it does not prevent Ras induced transformation in human HT1080, EJ30 or SW48 cells. Rapl also plays a critical role in regulation of normal morphogenesis in the eye disk and the ovary during embryo development. Recently, Rapl has been implicated in integrin-mediated . adhesion through RapL and modulation of the actin cytoskeleton through RIAM. The downstream effectors through which Rapl mediates its diverse effects are largely unknown. Screening the human genome based upon structural and functional information from the known effectors of Rapl such as RapL, RaIGDS, RIAM and Kritl, a series of potential effectors of Rapl have been identified. Using classical and modem biochemical approaches, it has been established that RASSFlA, a well-known tumour suppressor of human carcinogenesis, is a novel effector of RaplA - linking RaplA to microtubules. Moreover, Rapl association influences the effect of RASSFIA on microtubule behaviour. It is shown that interaction between RASSFIA and RaplA is modulated by RASSFIA phosphorylation. In silica analyses on RASSFIA predicted various putative phosphorylation sites and associated protein kinases within and outside the RA domain of RASSFIA. The sites S197 and S203 within the RA domain of RASSFIA were established as novel PKC phosphorylation sites by in vitro and in vivo assays. These two sites were, however, not responsible for the regulated interaction of RASSFIA with RaplA. This study provides important new insights into the signalling of RaplA indicating that regulated association with RASSFIA links RaplA to microtubules and that the RaplA-RASSFIA interaction plays a role in microtubule dynamics.

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