Yes / Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell.

Os mastócitos participam do sistema imunológico liberando mediadores farmacologicamente ativos. A principal via de ativação dos mastócitos é através do receptor de alta afinidade para a imunoglobulina E (FcRI). A ativação dos mastócitos via FcRI culmina com a liberação de mediadores. A enzima PLD atua sobre fosfolipídios hidrolisando a fosfatidilcolina em ácido fosfatídico e colina. A PLD é ativada após o estímulo via FcRI e possui um papel importante na transdução do sinal em mastócitos. Existem duas isoformas da enzima PLD, a PLD1 e a PLD2 que são expressas, diferentemente, de acordo com o tipo celular. Ambas as isoformas podem estar expressas numa mesma célula, apenas uma ou nenhuma. Neste estudo foram utilizadas células RBL-2H3 transfectadas para a super expressão PLD2 nas formas catalítica ativa (CA) e inativa (CI). O papel da PLD2 foi examinado nestas células com o objetivo de elucidar sua atuação no processo de secreção incluindo o aparelho de Golgi e os grânulos secretores. As células CA e CI possuem maior atividavidade de -hexosaminidase total, porém quando estimuladas mostram uma deficiência na liberação desta enzima, quando comparadas com as células selvagens. A PLD2 nas células CA, CI, VET e RBL-2H3 está localizada no citosol, sendo abundante na região justanuclear, principalmente nas células CI, sugerindo uma associação com o aparelho de Golgi. A dupla marcação com o mAb AA4, que imunomarca gangliosídeos derivados do GD1b da membrana plasmática e com anti-PLD2, mostrou que esta enzima não se localiza na membrana plasmática. A dupla marcação com anti-PLD2 e anti-GM130 mostrou que as áreas de maior concentração da PLD2 se co-localizam com o aparelho de Golgi, especialmente nas células CI. A marcação com anti-GM130 e os experimentos com microscopia eletrônica de transmissão mostraram que o aparelho de Golgi está organizado nas células CA e desorganizado nas células CI, onde se encontra disperso no citoplasma. Ainda, as células CI expressam menos GM130 em comparação com as demais linhagens celulares. Quando a produção de PA pela PLD está inibida pelo 1-Butanol, as células CA apresentam as mesmas características fenotípicas das células CI. A incubação das CI com PA resulta na reestruturação do aparelho de Golgi. A manutenção estrutural do aparelho de Golgi, também está relacionada com os microtúbulos. Nas células CI o centro organizador de microtúbulos é dificilmente identificado. Os microtúbulos nas células CI são desordenados em comparação com as demais linhagens celulares. Estes resultados mostram que a produção de PA pela PLD2 é importante na organização de microtúbulos e na manutenção da estrutura do aparelho de Golgi. As alterações celulares relacionadas com os microtúbulos e o aparelho de Golgi afetam o processo secretor nestas células e, provavelmente, em outros tipos de células secretoras. Estes achados poderão levar a novas estratégias terapêuticas para controlar a liberação de mediadores durante processos alérgicos e inflamatórios. / Mast cells are components of the immune system that liberate a wide variety of pharmacologically active mediators. The principle method of activating mast cells is through the high affinity receptor for IgE (FcRI). This activation then culminates with the release of mediators. Phospholipase D (PLD) acts on phospholipids, hydrolyzing phosphatidylcholine to phosphatidic acid (PA) and choline. PLD is activated following stimulation via FcRI and plays an important role in signal transduction in mast cells. PLD has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type where none, one or both may be expressed. RBL-2H3 cells, a mast cell line, transfected to super express catalytically active (CA) and inactive (CI) forms of PLD2 were used in the present study. The role of PLD2 was examined in these cells in order to clarify the action of PLD2 in the secretory process. Although the CA and CI cells posses a greater total -hexosaminidase activity, when stimulated these cells release less -hexosaminidase than cells transfected with empty vector or wild type RBL-2H3 cells. In all cell lines, PLD2 was dispersed throughout the cytoplasm with a concentration in the juxtanuclear region suggesting an association of PLD2 with the Golgi apparatus. Double labeling with anti-PLD2 and mAb AA4, which recognizes gangliosides derived from GD1b on the plasma membrane, showed that PLD2 was not associated with the plasma membrane. When the cells were double labeled with anti-PLD2 and anti-GM130, which labels the cis-Golgi saccules, PLD2 does colocalize with the Golgi apparatus, especially in CI cells. Labeling with anti-GM130 alone as well as experiments employing transmission electron microscopy revealed that the Golgi apparatus is well organized in the CA cells, but is disorganized and dispersed in the cytoplasm in the CI cells. By Western Blotting, the CI cells also expressed less GM130 than the other cell lines. When the production of PA by PLD2 was inhibited by 1-Butanol, the Golgi apparatus of the CA cells presented the same phenotypic characteristics as that of the CI cells. Conversely, incubation of the CI cells with PA resulted in the reorganization of the Golgi apparatus. The structural maintenance of the Golgi apparatus is also related to microtubules. In the CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized in the cytoplasm as compared to the other cell lines. These results show that the production of PA by PLD2 is important in the arrangement of the microtubules and in maintaining the structure of the Golgi apparatus. Alterations in the distribution of the microtubules and the structure of the Golgi apparatus in the CI cells affect the secretory process in these cells, and such alterations may affect the secretory process in other cell types as well. The findings presented here may lead to new therapeutic strategies to control the production and release of mediators during allergic and inflammatory processes.

It is known that muscles possess both intrinsic and reflexive responses to stretch, both of which have been studied extensively. While much is known about heterogenic and autogenic reflexes during XER, these have not been well characterized during locomotion. In this study, we mapped the distribution of autogenic and heterogenic feedback in hindlimb extensor muscles using muscle stretch in the spontaneously locomoting premammillary decerebrate cat. We used natural stimulation and compared stretch-evoked force responses obtained during locomotion with those obtained during XER. The goal was to ascertain whether feedback was modulated between the two states. We found that heterogenic feedback pathways, particularly those emanating from MG, remained inhibitory during locomotion while autogenic feedback specifically in MG increases in gain. Furthermore, increases in MG gain were due to force-dependent mechanisms. This suggests that rather than an abrupt transition from inhibition to excitation with changes in motor tasks, these pathways coexist and contribute to maintaining interjoint coordination. Increases in autogenic gain provide a localized loading reflex to contribute to the completion of the movement. The results of these experiments are clinically significant, particularly for the rehabilitation of spinal cord injured patients. To effectively administer treatment and therapy for patients with compromised spinal reflexes, a complete understanding of the circuitry is required.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references.

Thesis (Ph. D.)--Lunds universitet, 2005. / Title from title screen. Description based on contents viewed May 20, 2005. Includes bibliographical references (p. [36]-[48]).

Os mastócitos participam do sistema imunológico liberando mediadores farmacologicamente ativos. A principal via de ativação dos mastócitos é através do receptor de alta afinidade para a imunoglobulina E (FcRI). A ativação dos mastócitos via FcRI culmina com a liberação de mediadores. A enzima PLD atua sobre fosfolipídios hidrolisando a fosfatidilcolina em ácido fosfatídico e colina. A PLD é ativada após o estímulo via FcRI e possui um papel importante na transdução do sinal em mastócitos. Existem duas isoformas da enzima PLD, a PLD1 e a PLD2 que são expressas, diferentemente, de acordo com o tipo celular. Ambas as isoformas podem estar expressas numa mesma célula, apenas uma ou nenhuma. Neste estudo foram utilizadas células RBL-2H3 transfectadas para a super expressão PLD2 nas formas catalítica ativa (CA) e inativa (CI). O papel da PLD2 foi examinado nestas células com o objetivo de elucidar sua atuação no processo de secreção incluindo o aparelho de Golgi e os grânulos secretores. As células CA e CI possuem maior atividavidade de -hexosaminidase total, porém quando estimuladas mostram uma deficiência na liberação desta enzima, quando comparadas com as células selvagens. A PLD2 nas células CA, CI, VET e RBL-2H3 está localizada no citosol, sendo abundante na região justanuclear, principalmente nas células CI, sugerindo uma associação com o aparelho de Golgi. A dupla marcação com o mAb AA4, que imunomarca gangliosídeos derivados do GD1b da membrana plasmática e com anti-PLD2, mostrou que esta enzima não se localiza na membrana plasmática. A dupla marcação com anti-PLD2 e anti-GM130 mostrou que as áreas de maior concentração da PLD2 se co-localizam com o aparelho de Golgi, especialmente nas células CI. A marcação com anti-GM130 e os experimentos com microscopia eletrônica de transmissão mostraram que o aparelho de Golgi está organizado nas células CA e desorganizado nas células CI, onde se encontra disperso no citoplasma. Ainda, as células CI expressam menos GM130 em comparação com as demais linhagens celulares. Quando a produção de PA pela PLD está inibida pelo 1-Butanol, as células CA apresentam as mesmas características fenotípicas das células CI. A incubação das CI com PA resulta na reestruturação do aparelho de Golgi. A manutenção estrutural do aparelho de Golgi, também está relacionada com os microtúbulos. Nas células CI o centro organizador de microtúbulos é dificilmente identificado. Os microtúbulos nas células CI são desordenados em comparação com as demais linhagens celulares. Estes resultados mostram que a produção de PA pela PLD2 é importante na organização de microtúbulos e na manutenção da estrutura do aparelho de Golgi. As alterações celulares relacionadas com os microtúbulos e o aparelho de Golgi afetam o processo secretor nestas células e, provavelmente, em outros tipos de células secretoras. Estes achados poderão levar a novas estratégias terapêuticas para controlar a liberação de mediadores durante processos alérgicos e inflamatórios. / Mast cells are components of the immune system that liberate a wide variety of pharmacologically active mediators. The principle method of activating mast cells is through the high affinity receptor for IgE (FcRI). This activation then culminates with the release of mediators. Phospholipase D (PLD) acts on phospholipids, hydrolyzing phosphatidylcholine to phosphatidic acid (PA) and choline. PLD is activated following stimulation via FcRI and plays an important role in signal transduction in mast cells. PLD has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type where none, one or both may be expressed. RBL-2H3 cells, a mast cell line, transfected to super express catalytically active (CA) and inactive (CI) forms of PLD2 were used in the present study. The role of PLD2 was examined in these cells in order to clarify the action of PLD2 in the secretory process. Although the CA and CI cells posses a greater total -hexosaminidase activity, when stimulated these cells release less -hexosaminidase than cells transfected with empty vector or wild type RBL-2H3 cells. In all cell lines, PLD2 was dispersed throughout the cytoplasm with a concentration in the juxtanuclear region suggesting an association of PLD2 with the Golgi apparatus. Double labeling with anti-PLD2 and mAb AA4, which recognizes gangliosides derived from GD1b on the plasma membrane, showed that PLD2 was not associated with the plasma membrane. When the cells were double labeled with anti-PLD2 and anti-GM130, which labels the cis-Golgi saccules, PLD2 does colocalize with the Golgi apparatus, especially in CI cells. Labeling with anti-GM130 alone as well as experiments employing transmission electron microscopy revealed that the Golgi apparatus is well organized in the CA cells, but is disorganized and dispersed in the cytoplasm in the CI cells. By Western Blotting, the CI cells also expressed less GM130 than the other cell lines. When the production of PA by PLD2 was inhibited by 1-Butanol, the Golgi apparatus of the CA cells presented the same phenotypic characteristics as that of the CI cells. Conversely, incubation of the CI cells with PA resulted in the reorganization of the Golgi apparatus. The structural maintenance of the Golgi apparatus is also related to microtubules. In the CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized in the cytoplasm as compared to the other cell lines. These results show that the production of PA by PLD2 is important in the arrangement of the microtubules and in maintaining the structure of the Golgi apparatus. Alterations in the distribution of the microtubules and the structure of the Golgi apparatus in the CI cells affect the secretory process in these cells, and such alterations may affect the secretory process in other cell types as well. The findings presented here may lead to new therapeutic strategies to control the production and release of mediators during allergic and inflammatory processes.

Les molécules d’adhésion jonctionnelles JAM-B et JAM-C forment une paire récepteur/ligand impliquée dans la régulation de nombreux mécanismes biologiques dont l’inflammation, l’hématopoïèse et la spermatogénèse. Dans la moelle osseuse, l’interaction entre JAM-C et JAM-B, respectivement exprimée par les cellules souches hématopoïétiques (CSH) et les cellules stromales, joue un rôle dans la rétention et la quiescence des CSH. Dans le testicule, JAM-C participe à la polarisation des spermatides en différenciation en interagissant avec JAM-B exprimée par les cellules de Sertoli. GRASP55 (Golgi ReAssembly and Stacking Protein of 55 kDa), identifiée au laboratoire comme un interacteur intracellulaire des protéines JAMs, est une protéine de l’appareil de Golgi participant à l’architecture et la dynamique de celui-ci ainsi qu’au transport protéique non-conventionnel.Le but de mon travail de thèse a été d’étudier le rôle de GRASP55 in vivo par des approches génétiques et pharmacologiques. Nous avons ainsi pu mettre en évidence que l’expression de GRASP55 par la spermatide ronde permet la localisation polarisée de JAM-C et le déroulement correct de la spermatogénèse. A contrario, GRASP55 n’est pas essentiel à l’hématopoïèse en conditions basales ou de stress. Toutefois, la délétion de GRASP-55 dans les cellules leucémiques diminue le progression de la pathologie in vivo. Ces résultats montrent un rôle non redondant de GRASP55 dans la spermatogenèse et la prolifération de cellules leucémiques et ouvrent des pistes possibles pour un ciblage thérapeutique de GRASP55 en hématologie. / The junctional adhesion molecules JAM-B and JAM-C form a receptor / ligand pair involved in regulation of many biological mechanisms including inflammation, hematopoiesis and spermatogenesis. In the bone marrow, the interaction between JAM-C and JAM-B, expressed by hematopoietic stem cells (HSC) and stromal cells respectively, is involved in HSC retention and quiescence. Similarly, in the testis, JAM-C participates in the polarization of differentiated spermatids by interacting with JAM-B expressed by Sertoli cells. GRASP55 (Golgi ReAssembly and Stacking Protein of 55 kDa), identified in our laboratory as a new intracellular interactor of JAM, is a Golgi apparatus protein involved in Golgi architecture and dynamics as well as unconventional secretion.The aim of my thesis was to study the role of GRASP55 in vivo by genetic and pharmacological approaches. We demonstrate that GRASP55 expression by round spermatid allows polarized localization of JAM-C and the correct course of the spermatogenesis. In contrast, GRASP55 is not essential for hematopoiesis in basal or stress conditions. However, deletion of GRASP-55 in leukemic cells decreases the progression of the pathology in vivo. These results show a non-redundant role of GRASP55 in the spermatogenesis and proliferation of leukemic cells and allow us to consider GRASP55 as a potential target in hematology.

CONTEXTE: Les " Type I Congenital disorders of glycosylation " (CDG-I) comportent des déficits de biosynthèse de l'oligosaccharide lié au dolichol (DLO) qui est nécessaire pour la N-glycosylation des protéines. Ces déficits induisent : 1) une hypoglycosylation des protéines qui serait à l'origine de la pathologie ; et 2) une accumulation de DLO tronqués à partir desquels, par un mécanisme encore inconnu, des structures oligosaccharidiques libres phosphorylées (OSP) sont générées dans le cytosol. Afin de comprendre le rôle de ce processus dans le CDG, il était donc nécessaire de caractériser l'activité qui est à l'origine des OSP.RESULTATS: J'ai caractérisé biochimiquement une DLO diphosphatase (DLODP) qui génère des OSP et du dolichol phosphate à partir de DLO. L'activité DLODP co-fractionne avec un marqueur de l'appareil de Golgi (AG) mais pas avec les enzymes réticulaires qui utilisent le dolichol phosphate. Cette localisation inattendue de DLODP m'a conduit à étudier la génération des OSP dans les cellules en utilisant la bréfeldine A (BFA) qui fusionne l'AG avec le RE. La BFA ne modifie pas les taux de DLO tronqués ni ceux des OSP cytoplasmiques dans un modèle cellulaire de CDG-I. Cependant, dans ces cellules et dans les cellules témoins, la BFA induit une forte augmentation des OSP dans le système endomembranaire à partir de DLO non-tronqués.CONCLUSION: L'identification de différents pools d'OSP, topologiquement distincts et pouvant être modulés de façon indépendante, révèle la multiplicité des mécanismes pour la génération d'OSP et suggère que la DLODP Golgienne n'est pas forcément l'enzyme responsable de la génération des OSP dans le contexte de CDG-I. / BACKGROUND: Type I congenital disorders of glycosylation (CDG-I) are caused by genetic defects in the biosynthetic pathway for the dolichol-linked oligosaccharide (DLO) that is required for protein N-glycosylation. These mutations result in the accumulation of truncated DLO and protein hypoglycosylation. Although protein hypoglycosylation is thought to be the main pathogenic factor in CDG-I, the role of truncated DLO intermediates in cellular homeostasis is not clear. Truncated DLO intermediates are known to give rise to cytoplasmic oligosaccharyl phosphates (OSP) by an uncharacterized mechanism. To understand this DLO editing process biochemical and molecular characterization of the activity that generate OSP is needed.RESULTS: I biochemically characterized a DLO diphosphatase (DLODP) that generates OSP and dolichol phosphate from DLO. Subcellular fractionation of mouse liver homogenates demonstrated a microsomal activity that co-distributes with a Golgi apparatus (GA) marker but not with endoplasmic reticulum (ER)-situated dolichol phosphate utilizing enzymes. This unexpected localization of DLODP prompted me to study OSP generation in cells using brefeldin A (BFA), which fuses the GA with the ER. BFA did not affect the levels of truncated DLO or cytoplasmic OSP, present in a cellular model of CDG-I. However, in these, and control cells, BFA caused striking increases of OSP within the endomembrane system. CONCLUSION: the identification of topologically distinct, independently modulated, OSP pools indicates multiple mechanisms for OSP generation and suggest that the GA-situated DLODP may not be the enzyme responsible for OSP generation in CDG-I.

Les septines sont une famille de protéines GTPases qui peuvent former des structures d'ordre supérieur, comme les filaments et les anneaux, et capables de se lier avec les membranes cellulaires par leur interaction avec les phosphoinositides (PIs) via un domaine polybasique en N-terminal de leur domaine de liaison au GTP. Nous avons montré par une analyse transcriptomique réalisée en utilisant les données GSE14323 que la septine 9 est significativement surexprimée dans la cirrhose induite par le virus de l'hepatite C (VHC). Nos résultats montrent, ainsi, que la septine 9 induit l’augmentation en taille des gouttelettes lipidiques (GLs) par un mécanisme dépendant le phosphatidylinositol-5-phosphate et des microtubules. Nous avons montré, également, que cette voie de régulation des GLs est exploité par le VHC. De plus, nous avons montré que la septine 9 est impliquée dans la régulation de la morphologie de l’appareil Golgi et la mise en place de la polarité cellulaire par son interaction avec les phosphoinositides via deux domaines polybasiques. Ces résultats apportent une nouvelle compréhension du mécanisme moléculaire de l’interaction des septines avec les phosphoinositides et montrent pour la première fois l’importance de cette interaction dans des fonctions cellulaires de la septine 9. / Septins are a GTPases proteins family that can form high order structures such as filaments and rings, and able to bind cell membranes by interacting with phosphoinositides via a polybasic domain located at the N-terminal of their GTP binding domain. Here, We show by the transcriptomic analysis performed using the GSE14323 dataset that septin 9 is significantly upregulated in hepatitis C virus induced cirrhosis. Our findings show that septin 9 induce the lipid droplet growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV. In addition, we have shown that the septin 9 is involved in Golgi apparatus morphology regulation and cell polarity installation by interacting with phosphoinositides via two polybasic domains. These results provide new understanding of the molecular mechanism of septins interaction with the phosphoinositides and show its importance in septin 9 cellular functions shown for the first time.

Les protéines SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) sont essentielles pour la fusion membranaire. J'ai étudié chez Arabidopsis thaliana la SNARE Memb11 de l’appareil de Golgi qui intervient au début de la voie sécrétoire à l'interface Réticulum endoplasmique (RE)-appareil de Golgi. Dans les cellules de mammifères, l'orthologue de Memb11 (Membrine) est un « récepteur » potentiel de la GTPase Arf1 à la membrane golgienne. Cette dernière est impliquée dans le recrutement de la machinerie COPI nécessaire au transport rétrograde de l'appareil de Golgi vers le RE. Le but de ce travail était de déterminer si Memb11 pouvait interagir avec Arf1 dans les cellules végétales. Des anticorps dirigés contre la partie cytosolique de Memb11 ont été obtenus et ont été utilisés sur tissus végétaux pour réaliser des immunomarquages en microscopie électronique à transmission et des immunoprécipitations sur extraits de plantes. Il a été démontré que Memb11 est située au niveau de la membrane cis-golgienne et qu'elle co-immunoprécipite avec Arf1, suggérant ainsi que Arf1 peut interagir avec Memb11. J'ai confirmé l'interaction de Memb11 et Arf1 au niveau de l'appareil de Golgi par des expériences de BiFC (Bimolecular Fluorescence Complementation) in vivo. Cette interaction est spécifique puisque ni Memb12 (90% d'identité avec Memb11) ni Sec22 interagissent avec Arf1. Grâce à une approche de bioinformatique structurale, j'ai déterminé les régions de Memb11 (différentes de Memb12) qui pourraient être critiques pour l'interaction et j’ai commencé à tester in vivo les mutants correspondants par BiFC. En outre, des expériences d’immunoprécipitations avec des protéines recombinantes produites in vitro suggèrent que la forme d’Arf1 liée au GTP interagit avec Memb11. / The SNARE proteins (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) are critical for membrane fusion in the secretory pathway. I have studied the Golgi SNARE Memb11 in Arabidopsis thaliana cells. Memb11 is involved at the ER-Golgi interface. In mammalian cells, the ortholog of Memb11 (Membrin) is the potential “receptor” of the GTPase Arf1 in the Golgi membrane. This protein is involved for the recruitment of the COPI machinery, required for retrograde transport from the Golgi to the ER. The aim of this work was to determine whether Memb11 can interact with Arf1 in plant cells. Antibodies against the cytosolic part of Memb11 were obtained and were applied on plant tissues to perform immunolabeling by transmission electron microscopy and immunoprecipitation (IP) studies. It has been shown that Memb11 is located at the cis-Golgi and that it co-immunoprecipated with Arf1, suggesting that Arf1 may interact with Memb11. I confirmed the interaction of Memb11 and Arf1 at the Golgi by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was specific since neither Memb12 (90% identity with Memb11) nor Sec22 interacted with ARF1. Thanks to a structural bioinformatic approach, I determined the regions in Memb11 (different from Memb12) that could be critical for the interaction and started to test corresponding mutants in vivo by BiFC. In addition, IP experiments with recombinant proteins produced in vitro suggest that the GTP-bound form of ARF1 interacts with Memb11.