Plant cells are mostly immobile, therefore it is crucial for them to distinguish a direction of the signals coming into the cell and on the other hand they have to precisely target their own signals. To achieve this communication, plant cells use endomembrane system and secretory vesicles, which are recruited to the specific membrane domains. This ability is important for the plant defense against pathogenic microorganisms and it even forms a part of the innate plant immunity. Two complexes, the exocyst and SNARE, play a prominent role in the process of polarized secretion. In this work, we focused on a possible interaction between these two complexes in preinvasive defense and particularly, we studied the exocyst subunit EXO70B2 and SNARE protein SYP121. We obtained double mutant plants of EXO70B2 and SYP121 by utilizing the reverse genetics approach. These mutant plants did not show any obvious phenotype under standard conditions in comparison with Wt plants. However, we observed marked defects of secretory pathway in double mutant exo70B2/syp121 after infection by pathogenic fungi Blumeria graminis f. sp. hordei. Using histochemical staining, we described problems with the deposition of defensive papilla and secretion of haustorial encasement. We prove that these defects are not connected with...

Die in der vorliegenden Habilitationsschrift zusammengefaßten Publikationen stellen Untersuchungen zu zwei Themenschwerpunkten dar: 1. Verankerungsmechanismen von Membranproteinen der basolateralen und der apikalen Plasmamembrandomäne der Parietalzellen mit dem Membranzytoskelett und 2. die regulierte Fusion von zytoplasmatischen Vesikeln mit der apikalen Plasmamembran dieser Zellen. Die strukturell und molekular sehr unterschiedlich gestaltete apikale und basolaterale Membrandomäne der Parietalzellen sollte funktionell charakterisiert und die Mechanismen der Membranumbauvorgänge aufgeklärt werden, die nach Aktivierung der Zellen im apikalen Membrankompartiment ablaufen. Für die strukturelle Stabilität der basolateralen Domäne spielt wahrscheinlich die Verankerung von AE2 über das Verknüpfungsprotein Ankyrin mit dem Membranzytoskelett eine wichtige Rolle. Die apikale Membrandomäne der Parietalzellen kann in drei Kompartimente unterteilt werden. Die freie apikale Membran, die canalikuläre Membran und die Membranen der tubulären Vesikel. Entlang der freien apikalen und der canaliculären Plasmamembran kommen wie auf der basolateralen Seite die Zytoskelett-Proteine Actin und Spectrin vor. Nach unseren Untersuchungen könnte es während der Sekretionsphase zu einer temporären Verbindung von H+,K+-ATPase Molekülen mit dem Membranzytoskelett kommen. Diese Verbindung wird wahrscheinlich durch das Verknüpfungsprotein Ezrin vermittelt. Der Mechanismus des Fusionsvorgangs der tubulären Vesikel mit der canaliculären Membran war bisher nicht bekannt. In Parietalzellen konnten die neuronalen SNARE-Proteine Synaptobrevin 2, Syntaxin 1 und SNAP25 sowie das zur Familie der kleinen G-Proteine gehörende Protein Rab3A und die Regulatorproteine NSF und alpha/beta SNAP nachgewiesen werden. Das in Parietalzellen gefundene Verteilungsmuster der SNARE-Proteine entspricht nicht der klassischen Vorstellung einer heterotypischen Membranfusion, vielmehr entspricht diese Verteilung einer homotypischen Fusion, wie sie für Vakuolen in Hefezellen beschrieben wurde. Die Bedeutung der SNARE-Proteine für die Fusion der tubulären Vesikel mit der canaliculären Membran und damit für die Steigerung der HCl-Sekretion konnte durch Inkubation der Zellen mit Tetanus Neurotoxin (TeNt) gezeigt werden. Die Behandlung der Parietalzellen mit TeNt führte zum vollständigen Ausbleiben der, nach Stimulation mit cAMP bei Kontrollzellen beobachteten Erhöhung, der Säuresekretion / The publications summarized here cover two topics: 1. the anchorage mechanism of membrane proteins of the basolateral and the apical plasma membrane with the membrane cytoskeleton of parietal cells and 2. the regulated fusion of cytoplasmic vesicles with the apical plasma membrane of these cells. It was the aim of these studies to characterize the structural and molecular differences between the apical and basolateral membrane domains in parietal cells. Moreover the mechanisms involved in membrane traffic within the apical membrane compartment following stimulation were investigated. We found that anchorage of AE2 with the membrane cytoskeleton through the linkage protein ankyrin seems to be important for the stability of the basolateral membrane. The apical membrane domain of parietal cells can be subdivided into three compartments. The free apical membrane, the canalicular membrane and the tubulovesicular membrane. The cytoskeletal proteins spectrin and actin can be found at the basolateral, the free apical and the canalicular membrane. We have shown that the H+K+-ATPase molecules appear to be temporary linked to the membrane cytoskeleton during acid-secretion. This contact is most likely mediated by the linker-protein ezrin. Until now the mechanism of fusion of the tubulovesicles with the canalicular membrane was unknown. In parietal cells the neuronal SNARE-proteins synaptobrevin 2, Syntaxin 1, SNAP25, the small G-protein rab3A, and the regulatory proteins NSF and alpha/beta-SNAP were detected. The subcellular distribution of these proteins does not support the notion of a neuron-like heterotypic fusion. Instead it shows similarity with the homotypic fusion process of vacuoles in yeast. The importance of SNARE-proteins for the fusion of tubulovesicles with the canalicular membrane and, by consequence also for the increase of acid-secretion was shown by incubation of the cells with tetanus neurotoxin (TeNt). The measurable increase of acid secretion by parietal cells after stimulation with c-AMP was inhibited completely through an incubation with TeNt.

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants containing phosphomimetics showed enhanced initial fusion rates. These results, combined with previous work in vivo, confirm that SNAP-23 phosphorylation is involved in regulating membrane fusion, and that IKK-mediated signaling contributes to platelet exocytosis. To address the role(s) of acylation, I sought to determine how syntaxin-11 and SNAP-23 are associated with plasma membrane. Using metabolic labeling, I showed that both proteins contain thioester-linked acyl groups which turn over in resting cells. Mass spectrometry mapping showed that syntaxin-11 is modified on C275, 279, 280, 282, 283 and 285, while SNAP-23 is modified on C79, 80, 83, 85, and 87. To probe the effects of acylation, I measured ADP/ATP release from platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. Cerulenin pretreatment inhibited t-SNARE acylation and platelet function while palmostatin B had no effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state of platelet proteins is important for their function. Thus my work indicates that the enzymes controlling protein acylation could be valuable targets for modulating platelet exocytosis in vivo.

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the identity of the functional syntaxin has been controversial. Previous studies, using anti-syntaxin antibodies in permeabilized platelets, suggested roles for both syntaxin-2 and -4. These conclusions were experimentally tested using platelets from syntaxin knockout mice and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient that lacks syntaxin-11. Platelets from syntaxin-2 and syntaxin-4 single or double knockout mice had no significant secretion defect. However, platelets from the FHL4 patient had a robust defect, though their morphology, activation, and cargo levels appeared normal. Semi-quantitative western blotting showed that syntaxin-11 is the most abundant syntaxin in both human and murine platelets. Co-immunoprecipitation experiments showed that syntaxin-11 forms SNARE complexes with VAMP-8 and SNAP-23. These data conclusively demonstrate that syntaxin-11, but not syntaxin-2, or -4, is required for platelet exocytosis. We also show that a syntaxin binding protein, tomosyn-1, is important for platelet exocytosis and hemostasis. Tomosyn-1 was identified from platelet extracts using affinity chromatography, RT-PCR analysis, and western blotting analysis. Tomosyn-1 was co-immunoprecipitated with syntaxin-11/SNAP-23 from both resting and activated platelet extracts. Platelets from tomosyn-1-/- mice displayed a secretion defect, but their morphology and activation appeared normal. Tomosyn-1-/- mice showed impaired thrombus formation in two different injury models. Given the importance of platelet secretion to hemostasis, it is hoped that the insights gained from these studies in this dissertation will help to identify new and more valuable therapeutic targets to control clot formation.

Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly. Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane. To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.

No description available.

Yersinia pseudotuberculosis appartient à la famille des Enterobacteriaceae et peut être responsable de syndromes articulaires et digestifs. Au cours de la colonisation de l’hôte, une minorité des bactéries va, en plus de l’étape de multiplication extracellulaire présenter une phase de réplication intracellulaire dans les macrophages. Une partie des Y. pseudotuberculosis va se répliquer dans les macrophages en usurpant la voie de l’autophagie, afin de créer une niche réplicative au sein des autophagosomes bloqués dans leur maturation. Le trafic membranaire associé à l’infection de Y. pseudotuberculosis reste à ce jour peu caractérisé. Dans un premier temps, nous avons observé que lors de l’infection d’une cellule épithéliale par Y. pseudotuberculosis, la vacuole bactérienne est associée avec le marqueur des autophagosomes, la protéine LC3 mais de façon surprenante cette vacuole ne présente pas deux mais une membrane unique. Par ailleurs, nous avons montré que les protéines SNARE jouent un rôle majeur au cours du trafic intracellulaire de Y. pseudotuberculosis. VAMP3 et VAMP7 sont recrutées de manière séquentielle au niveau de la vacuole de Y. pseudotuberuclosis. VAMP7 va participer au recrutement de LC3 au niveau de la vacuole bactérienne et nous proposons que VAMP3 est un des constituants du check-point permettant l’adressage de la bactérie vers des vacuoles présentant une ou de multiple membranes positives pour LC3. Par la suite, nous nous sommes intéressés à la caractérisation des protéines de la voie autophagique et des endosomes, recrutées au niveau de la vacuole bactérienne à membrane unique et positive pour LC3. Nous avons mis en évidence que les protéines impliquées dans la formation de l’autophagosome et les marqueurs des endosomes précoces sont recrutées au niveau de la vacuole contenant Y. pseudotuberculosis. Cette vacuole positive pour LC3 va en suite acquérir les marqueurs des endosomes tardifs et du lysosome mais n’est pas acidifiée. En outre, nous avons initié des travaux sur un criblage en haut contenu afin d’identifier les partenaires des protéines SNARE et leurs rôles dans le trafic intracellulaire de Y. pseudotuberuclosis. Ces travaux démontrent l’importance de l’analyse de l’ultrastructure des compartiments positifs pour LC3. Ils illustrent comment la bactérie s’adapte à son environnement pour établir sa niche réplicative. Ils présentent enfin l’importance de la régulation de l’autophagie avec la première mise en évidence d’un check-point entre deux voies de compartimentation positives pour LC3 mais morphologiquement différentes. / Yersinia pseudotuberculosis is a member of the Enterobacteriaceae family. In human, Y. pseudotuberculosis infection is responsible for enteric and, in rare cases, erythema nodosum. During host colonization, a minor part of Y. pseudotuberculosis presents an intracellular replication step. Y. pseudotuberculosis can replicate inside macrophages by hijacking the autophagy pathway. The bacteria are able to block autophagosome maturation by acidification impairment, which allows to create a replicative niche. The membrane traffic during internalization of Yersinia remains poorly characterized. First, we highlighted that in epithelial cells, Y. pseudotuberculosis replicates mainly in vacuoles positive for LC3, a hallmark of autophagy. Surprisingly, this LC3-positive-vacuole presents only single limiting membrane. Second, we showed that SNARE proteins play a role in Y. pseudotuberculosis intracellular traffic. VAMP3 and VAMP7 are sequentially recruited to Yersinia-containing vacuoles (YCVs). VAMP7 is involved in the LC3 recruitment to YCVs with single- and double-membrane. We proposed that VAMP3 is a component of the molecular checkpoint for bacterial commitment to either single- or double-membrane LC3-positive pathway. Third, we characterized the traffic of endosomal proteins recruited to LC3-positive-YCV with single membrane in epithelial cells. We showed that markers of early endosome and proteins involved in autophagosome formation, are recruited to YCVs during the early stage of infection. Then, the vacuole acquire late endosomal and lysosomal proteins but acidification is not observed. Finally, we initiated a high-content screening approach for the identification of SNARE partners.Overall this work illustrates the importance of LC3-positive compartment ultrastructure analysis. Our result demonstrate how bacterial subvert the molecular machinery of the host in order to create a replicative niche. Finally, we present the importance of autophagy regulation by highlighting for the first times the existence of a molecular checkpoint between two LC3-positive vacuoles with different morphologies

No description available.

The development of computer algorithms for music instrument identification and parameter extraction in digital audio signals is an active research field. A musician can listen to music and instantly identify different instruments and the timbres produced by various playing techniques. Creating software to allow computers to do the same is much more challenging. This thesis will use digital signal processing and machine learning techniques to differentiate snare drum timbres produced by different stroke positions and stroke techniques.

No description available.