Academic literature on the topic 'Membrane contact site'
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Journal articles on the topic "Membrane contact site"
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Westermann, Benedikt. "The mitochondria–plasma membrane contact site." Current Opinion in Cell Biology 35 (August 2015): 1–6. http://dx.doi.org/10.1016/j.ceb.2015.03.001.
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Bean, Björn D. M., Samantha K. Dziurdzik, Kathleen L. Kolehmainen, Claire M. S. Fowler, Waldan K. Kwong, Leslie I. Grad, Michael Davey, Cayetana Schluter, and Elizabeth Conibear. "Competitive organelle-specific adaptors recruit Vps13 to membrane contact sites." Journal of Cell Biology 217, no. 10 (July 17, 2018): 3593–607. http://dx.doi.org/10.1083/jcb.201804111.
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The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13–Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes.
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Gipson, Preeti, Yoshiyuki Fukuda, Radostin Danev, Ying Lai, Dong-Hua Chen, Wolfgang Baumeister, and Axel T. Brunger. "Morphologies of synaptic protein membrane fusion interfaces." Proceedings of the National Academy of Sciences 114, no. 34 (July 24, 2017): 9110–15. http://dx.doi.org/10.1073/pnas.1708492114.
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Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.
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Pfanner, Nikolaus, Martin van der Laan, Paolo Amati, Roderick A. Capaldi, Amy A. Caudy, Agnieszka Chacinska, Manjula Darshi, et al. "Uniform nomenclature for the mitochondrial contact site and cristae organizing system." Journal of Cell Biology 204, no. 7 (March 31, 2014): 1083–86. http://dx.doi.org/10.1083/jcb.201401006.
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The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.
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Mannella, C. A., K. F. Buttle, K. A. O‘Farrell, A. Leith, and M. Marko. "Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 966–67. http://dx.doi.org/10.1017/s0424820100167299.
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Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.
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Rinnerthaler, G., B. Geiger, and J. V. Small. "Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules." Journal of Cell Biology 106, no. 3 (March 1, 1988): 747–60. http://dx.doi.org/10.1083/jcb.106.3.747.
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We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.
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Quon, Evan, Aleksa Nenadic, Mohammad F. Zaman, Jesper Johansen, and Christopher T. Beh. "ER-PM membrane contact site regulation by yeast ORPs and membrane stress pathways." PLOS Genetics 18, no. 3 (March 3, 2022): e1010106. http://dx.doi.org/10.1371/journal.pgen.1010106.
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In yeast, at least seven proteins (Ice2p, Ist2p, Scs2/22p, Tcb1-Tcb3p) affect cortical endoplasmic reticulum (ER) tethering and contact with the plasma membrane (PM). In Δ-super-tether (Δ-s-tether) cells that lack these tethers, cortical ER-PM association is all but gone. Yeast OSBP homologue (Osh) proteins are also implicated in membrane contact site (MCS) assembly, perhaps as subunits for multicomponent tethers, though their function at MCSs involves intermembrane lipid transfer. Paradoxically, when analyzed by fluorescence and electron microscopy, the elimination of the OSH gene family does not reduce cortical ER-PM association but dramatically increases it. In response to the inactivation of all Osh proteins, the yeast E-Syt (extended-synaptotagmin) homologue Tcb3p is post-transcriptionally upregulated thereby generating additional Tcb3p-dependent ER-PM MCSs for recruiting more cortical ER to the PM. Although the elimination of OSH genes and the deletion of ER-PM tether genes have divergent effects on cortical ER-PM association, both elicit the Environmental Stress Response (ESR). Through comparisons of transcriptomic profiles of cells lacking OSH genes or ER-PM tethers, changes in ESR expression are partially manifested through the induction of the HOG (high-osmolarity glycerol) PM stress pathway or the ER-specific UPR (unfolded protein response) pathway, respectively. Defects in either UPR or HOG pathways also increase ER-PM MCSs, and expression of extra “artificial ER-PM membrane staples” rescues growth of UPR mutants challenged with lethal ER stress. Transcriptome analysis of OSH and Δ-s-tether mutants also revealed dysregulation of inositol-dependent phospholipid gene expression, and the combined lethality of osh4Δ and Δ-s-tether mutations is suppressed by overexpression of the phosphatidic acid biosynthetic gene, DGK1. These findings establish that the Tcb3p tether is induced by ER and PM stresses and ER-PM MCSs augment responses to membrane stresses, which are integrated through the broader ESR pathway.
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Balla, Tamas, Yeun Ju Kim, Alejandro Alvarez-Prats, and Joshua Pemberton. "Lipid Dynamics at Contact Sites Between the Endoplasmic Reticulum and Other Organelles." Annual Review of Cell and Developmental Biology 35, no. 1 (October 6, 2019): 85–109. http://dx.doi.org/10.1146/annurev-cellbio-100818-125251.
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Phospholipids are synthesized primarily within the endoplasmic reticulum and are subsequently distributed to various subcellular membranes to maintain the unique lipid composition of specific organelles. As a result, in most cases, the steady-state localization of membrane phospholipids does not match their site of synthesis. This raises the question of how diverse lipid species reach their final membrane destinations and what molecular processes provide the energy to maintain the lipid gradients that exist between various membrane compartments. Recent studies have highlighted the role of inositol phospholipids in the nonvesicular transport of lipids at membrane contact sites. This review attempts to summarize our current understanding of these complex lipid dynamics and highlights their implications for defining future research directions.
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Rassow, J., B. Guiard, U. Wienhues, V. Herzog, F. U. Hartl, and W. Neupert. "Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites." Journal of Cell Biology 109, no. 4 (October 1, 1989): 1421–28. http://dx.doi.org/10.1083/jcb.109.4.1421.
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Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.
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Bohnert, Maria, and Maya Schuldiner. "Stepping outside the comfort zone of membrane contact site research." Nature Reviews Molecular Cell Biology 19, no. 8 (May 15, 2018): 483–84. http://dx.doi.org/10.1038/s41580-018-0022-1.
Full textDissertations / Theses on the topic "Membrane contact site"
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Nicolas, William. "Understanding plasmodesmata membrane organization and the control of cell-to-cell connectivity in plants." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0213.
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La communication intercellulaire est essentielle pour le développement et la survie d'organismes multicellulaires. Dans le règne végétale, une des voies privilégiée pour la communication intercellulaire est la voie symplastique qui implique des canaux aux dimensions nanométriques connectant les cellules entre elles, leur permettant d'échanger directement photo-assimilats, miARN, protéines, oligoéléments etc. Observés pour la première fois en 1880 par le botaniste autrichien Eduard Tangl (Tangl 1880; Kohler & Carr 2006), ils ont longtemps été considérés comme de simples trous perméables permettant la diffusion de matériel cellulaire (Lee & Lu 2011; Oparka & Roberts 2001). Etant donné leurs taille nanoscopique, ce n'est que dans les années 1960, avec la démocratisation de la Microscopie Électronique en Transmission (MET) qui permet d'atteindre , que les premiers modèles ultrastructuraux sont établis (Lopez-Saez 1965; Robards 1970). Ils font état d'un canal d'environ 30 à 40 nm de diamètre avec un élément central cylindrique traversant le pore, appelé le desmotubule, connecté au Réticulum Endoplasmique des deux cellules (Figure 1 of our review Tilsner et al. 2016). Dans les années 1980 notre compréhension des plasmodesmes a quelque peu évolué et nous savons maintenant que ces structures ne sont pas de simples trous mais des structures membranaires très spécialisées et régulées (Lucas & Lee 2004; Faulkner & Maule 2011; Furuta et al. 2012). Le modèle ultrastructural actuel découle de la congrégation d'études ultrastructurale, physiologiques et pharmacologiques plus ou moins anciennes dépeignant une structure morphologiquement très souple et changeant de conformation au cours du développement. Les plasmodesmes peuvent réguler leur ouverture/fermeture par la constriction de leurs extrémités grâce à l'accumulation entre la membrane plasmique et la paroi végétale d'un polymère de sucre, la callose qui va pousser la membrane plasmique contre le desmotubule et en obstruer les entrées. Cette modulation permettrait majoritairement de réguler les flux intercellulaires qui impliquent les plasmodesmes. Cependant nos connaissances sur les remaniements membranaires prenant place durant le développement des plasmodesmes et sur la régulation de leur perméabilité sont encore imparfaites.La microscopie électronique en transmission, malgré l'ancienneté de la technique, est l'une des plus résolutive, largement utilisée en biologie. Avec l'amélioration des techniques de préservation d'échantillons, notamment les cryo- méthodes, elle permet d'atteindre à l'heure actuelle des résolutions inférieures à 5 nm en condition contrastée et inclus en résine et peut descendre en dessous du nanomètre pour la cryo-microscopie. Ce potentiel permet aisément l'étude des sous-compartiments cellulaires de l'ordre du µm tel que mitochondries, chloroplastes, noyaux etc. (Frey et al. 2002) mais permet également l'étude ultrastructurale précise de structures de l'ordre de la dizaine de nm (Beck et al. 2007; Al-Amoudi et al. 2007).En revanche, dans son utilisation classique, la microscopie électronique ne permet pas d'accéder à la troisième dimension de l'espace, rendant difficile l'interprétation de structure à l'architecture quelque peu compliquée. En effet, les images produites ne sont que des projections en deux dimensions d'objets en trois dimensions. Cela a mené au développement de la tomographie électronique en transmission (Crowther et al. 1970), méthode basée sur un concept mathématiques formulé par Johann Radon au XIXe siècle. Ce n'est que dans les années 2000 que la tomographie électronique a pris un essor significatif grâce au couplage avec des méthodes d'automatisation informatiquesPlasmodesmata were first observed by Austrian botanist Eduard Tangl in 1880. He devoted himself to studying the anatomy and cytology of plants and his greatest discovery, of course, was the observation and first characterization of plasmodesmata (Tangl 1880, 1884 and 1885). Despite not having access to their ultrastructure, he observed thin striations (see front page engraving) between cotyledon cells of Strychnos nuxvomica and in the endosperm of seeds and described them as being conductive ducts. Already at the time, he was evoking the idea that these strands "unite them [the cells] to an entity of higher order", in other words formulating the first definition of a symplastic domain. lt is only in 1901 that Strasburger finally names these canals "plasmodesmata". His discovery led to a radical change in our conception of the plant entity and brought in new concepts such as the symplasm (Munch 1930) and transmembrane fluxes between cells, which are now being tackled with great interest by numerous research teams around the globe.Because of their size, plasmodesmata ultrastructure was not accessible until the advent of electron microscopy and they were long thought to be simple holes connecting plant cells one-another with no specific regulation. lt is only with the advent of electron microscopy and chemical fixation that botanists started to gain interest in this structure again. And even with these methods allowing the observation of structures down to several nanometers in size, there are still debates on the nature of the canal, its constituents and physiology (Lopez-Saez J. 1965, Robards A. 1970, Ding et al. 1992, Tilney et al. 1991, Overall and Gunning 1982, Schulz et al. 1995).Nowadays, with the advent of modern cryopreservation and three-dimensional electron tomography methods, great improvements are to be done in the understanding of the ultrastructure and physiology of these mysterious canals. More particularly by understanding the link between the membranous rearrangements taking place in these pores and the molecular transit regulation.My work has led us to view plasmodesmata as specialised Membrane Contact Sites (MCS). Hence, by analogy with MCS found in mammals, yeast and plants, this work embraces an original angle on the speculation of the composition and role of the desmotubule-plasma-membrane tethering complex. The work produced during my thesis allowed me to contribute to the publication of one review and two articles, which will constitute the introduction and two main sub-sections of the results chapter, respectively. The introductory review has been published in 2016 in Annual Review of Plant Biology. The first one is still under reviewing at Nature Plant and the other has been published in The Plant Cell journal in April 2015
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Jamecna, Denisa. "Une région intrinsèquement désordonnée dans OSBP contrôle la géometrie et la dynamique du site de contact membranaire." Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4229/document.
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La protéine OSBP est un transporteur de lipides qui régule la distribution cellulaire du cholestérol. OSBP comprend un domaine PH, deux séquences « coiled coil », un motif FFAT (deux phénylalanines dans un environement acide), et un domaine de liaison de lipides (ORD) à son extrémité C-terminale. Le domaine PH interagit avec le PI(4)P et la petite protéine G Arf1-GTP au niveau du Golgi, alors que le motif FFAT interagit avec la protéine VAP-A, résidente du réticulum endoplasmique (RE). En liant simultanément tous ces déterminants, OSBP stabilise des sites de contact membranaire entre RE et Golgi, permettant ainsi un contre-échange cholestérol / PI(4)P par l'ORD. OSBP contient également une longue séquence N-terminale d’environ 80 aa, intrinsèquement désordonnée, composée principalement de glycine, proline et d'alanine. Nous démontrons que la présence de ce N-terminus désordonné augmente le rayon de Stoke de OSBP tronquée du domaine ORD, et limite sa densité d’association sur la membrane portant le PI(4)P. La protéine dépourvue du N terminus favorise l'agrégation symétrique des liposomes PI(4)P (mimant la membrane du Golgi) par les deux domaines PH du dimère OSBP, alors que la présence de la séquence désordonnée empêche cette association symétrique. De même, nous observons que la distribution d’OSBP sur la membrane de vésicules unilamellaires géantes (GUV) varie selon la présence ou l'absence du N-terminus. En présence de la séquence désordonnée, la protéine est répartie de manière homogène sur toute la surface du GUV, alors que la protéine sans N-terminal a tendance à s'accumuler à l'interface entre deux GUV de type Golgi. Cette accumulation locale ralentit fortement la mobilité de la protéine à l’interface. Un effet similaire du N-terminal sur la dynamique des protéines est observé lorsque l’association de membranes de type ER et Golgi est assuré par des protéines monomériques (dépourvue du coiled coil) en présence de Vap-A. Les résultats de nos expériences in vitro ont été confirmés en cellules vivantes, où la séquence intrinsèquement désordonnée contrôle le recrutement d’OSBP sur les membranes Golgiennes, sa mobilité et sa dynamique d’activité au cours des cycles de transfert de lipides. La plupart des protéines de la famille d’OSBP contiennent des séquences N-terminales de faible complexité, suggérant un mécanisme général de régulationOxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect
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Jamecna, Denisa. "Une région intrinsèquement désordonnée dans OSBP contrôle la géometrie et la dynamique du site de contact membranaire." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4229.
Full textAbstract:
La protéine OSBP est un transporteur de lipides qui régule la distribution cellulaire du cholestérol. OSBP comprend un domaine PH, deux séquences « coiled coil », un motif FFAT (deux phénylalanines dans un environement acide), et un domaine de liaison de lipides (ORD) à son extrémité C-terminale. Le domaine PH interagit avec le PI(4)P et la petite protéine G Arf1-GTP au niveau du Golgi, alors que le motif FFAT interagit avec la protéine VAP-A, résidente du réticulum endoplasmique (RE). En liant simultanément tous ces déterminants, OSBP stabilise des sites de contact membranaire entre RE et Golgi, permettant ainsi un contre-échange cholestérol / PI(4)P par l'ORD. OSBP contient également une longue séquence N-terminale d’environ 80 aa, intrinsèquement désordonnée, composée principalement de glycine, proline et d'alanine. Nous démontrons que la présence de ce N-terminus désordonné augmente le rayon de Stoke de OSBP tronquée du domaine ORD, et limite sa densité d’association sur la membrane portant le PI(4)P. La protéine dépourvue du N terminus favorise l'agrégation symétrique des liposomes PI(4)P (mimant la membrane du Golgi) par les deux domaines PH du dimère OSBP, alors que la présence de la séquence désordonnée empêche cette association symétrique. De même, nous observons que la distribution d’OSBP sur la membrane de vésicules unilamellaires géantes (GUV) varie selon la présence ou l'absence du N-terminus. En présence de la séquence désordonnée, la protéine est répartie de manière homogène sur toute la surface du GUV, alors que la protéine sans N-terminal a tendance à s'accumuler à l'interface entre deux GUV de type Golgi. Cette accumulation locale ralentit fortement la mobilité de la protéine à l’interface. Un effet similaire du N-terminal sur la dynamique des protéines est observé lorsque l’association de membranes de type ER et Golgi est assuré par des protéines monomériques (dépourvue du coiled coil) en présence de Vap-A. Les résultats de nos expériences in vitro ont été confirmés en cellules vivantes, où la séquence intrinsèquement désordonnée contrôle le recrutement d’OSBP sur les membranes Golgiennes, sa mobilité et sa dynamique d’activité au cours des cycles de transfert de lipides. La plupart des protéines de la famille d’OSBP contiennent des séquences N-terminales de faible complexité, suggérant un mécanisme général de régulationOxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect
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Petit, Jules. "Membrane Tethering in Plant Intercellular Communication : Structure-Function of Multiple C2 domains and Transmembrane Region Proteins (MCTP) at Plasmodesmata ER-PM Membrane Contact Site." Thesis, Bordeaux, 2022. https://tel.archives-ouvertes.fr/tel-03789611.
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La multicellularité chez les plantes repose sur une communication intercellulaire qui permette le transfert d'informations à travers l'entièreté de l'organisme. Chez les plantes terrestres, la route principale de ces “conversations cellulaires” est assurée par les plasmodesmes (PD), des canaux nanoscopiques qui traversent la paroi pecto-cellulosique. En effet, ces pores sont impliqués dans la circulation d'une très grande variété de molécules, comme des facteurs de transcription, de l'ARN, des hormones et des métabolites et ceci à tous les stades de la vie végétale, permettant réponses et adaptations à l'environnement. Les PD sont particuliers dans le sens où ils forment une continuité du réticulum endoplasmic (RE), de la membrane plasmique (MP) et du cytoplasme entre les cellules adjacentes. Leur architecture est singulière et consiste en un filament de RE, appelé desmotubule, entouré d'un tube de MP qui, lui, longe la paroi. Les PD sont actuellement décrits comme des sites de contact membranaire, du fait du fort accolement des membranes du RE et de la MP (2 à 10 nm) et de la présence de protéines de jonction qui connectent les deux organelles. Dans la présente étude, nous décrivons au niveau structural et fonctionnel plusieurs membres de la famille des MCTPs (protéine avec de multiples domaines C2 et une région transmembranaire) comme protéines assurant la jonction du RE et de la MP dans les PD. Nous démontrons que ces protéines possèdent les caractéristiques moléculaires nécessaires à l'interaction transitoire avec les lipides anioniques de la MP, via leurs domaines C2, ainsi qu'à l'induction de courbure membranaire au RE, via la région transmembranaire qui agit comme un domaine homologue aux protéines réticulons. Ces données nous ont permis de corréler la fonction des MCTPs à l'architecture et la biogenèse des PD et de réfléchir au rôle du RE à l'intérieur des PD. En conclusion, ce travail a fourni des résultats originaux qui placent les MCTPs comme des protéines centrales dans l'établissement de la structure fine des PD et des fonctions qui y sont associéesPlant multicellularity relies on intercellular communication in order to transmit information from cell to cell and throughout the entire plant body. In land plants, the major line for such cellular conversations is through plasmodesmata (PD) pores, which are nanoscopic membranous tunnels spanning the pecto-cellulosic cell wall. These pores are indeed involved in the transfer of a wide variety of molecules such as transcription factors, RNAs, hormones and metabolites during all stages of plant life, adaptation and responses to their environment. PD are singular amongst other types of intercellular junctions as they provide a direct continuity of the endoplasmic reticulum (ER), the plasma membrane (PM) and the cytosol between neighboring cells. Their architectural organization can be summarized as followed: a thin strand of constricted ER, called desmotubule, is encased in a tube of PM lining the cell wall. PD are seen as a specialized ER-PM membrane contact sites from the very close apposition (2 to 10 nm) of the ER and PM membranes and the presence of tethering elements bridging the two organelles. In this study, we describe the structural organization and function of several members of the MCTP (Multiple C2 domains and Transmembrane region Protein) family which act as ER-PM tethering elements at PD. We show that these proteins possess molecular features capable of transient interaction with anionic lipids of the PM, through their C2 domains, as well as ER membrane shaping, through their transmembrane region which presents homology to a reticulon domain. We further correlate MCTP function with PD architecture and biogenesis, and investigate on the role of the ER inside PD. Altogether, this work provides original data placing MCTPs as core PD proteins that appear to be crucial in the establishment of PD ultrastructure and associated functions
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Subra, Mélody. "VAP-A, un gymnaste moléculaire engagé dans les sites de contact membranaire." Electronic Thesis or Diss., Université Côte d'Azur, 2023. http://www.theses.fr/2023COAZ6013.
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VAP-A est un récepteur ancré à la surface du réticulum endoplasmique (RE) pour des centaines de protéines contenant un motif FFAT. Ses partenaires possèdent des structures et des fonctions très variées. VAP-A participe à la formation des sites de contact membranaire (MCSs) entre le RE et les autres organelles et ceci permet notamment le trafic non vésiculaire des lipides entre les membranes. Par exemple, la protéine de transfert de lipide OSBP interagit avec VAP aux MCSs RE/Golgi afin de transporter le cholestérol contre son gradient de concentration par contre-échange et hydrolyse de phosphatidylinositol-4-phoshate (PI4P). L'interaction entre le domaine Major-Sperm-Protein (MSP) de VAP-A et le motif FFAT de ses partenaires était déjà caractérisé. Cependant, la façon dont ce récepteur universel peut s'adapter à toutes ses cibles dans tous les MCSs, qui sont très différents en terme de géométrie et de stabilité était inconnue.Dans cette étude nous avons utilisé une approche pluridisciplinaire afin de démontrer que VAP-A contient deux régions intrinsèquement désordonnées (IDRs) qui fournissent à la protéine une flexibilité indispensable à son organisation fonctionnelle dans les MCSs. Nous avons montré qu'un mutant de VAP-A sans ses linkers flexibles possède une localisation subcellulaire restreinte aux MCSs RE/mitochondrie. Ce mutant ne peut donc pas soutenir l'activité d'OSBP et CERT aux MCSs RE/Golgi. En revanche, il interagit avec VPS13A et PTPIP51 à la mitochondrie et permet ainsi le transport de lipides qui contribuent au métabolisme des cardiolipines et à la fusion mitochondriale.Ces résultats indiquent que la flexibilité de VAP-A fournie par ses IDRs, joue un rôle clé pour assurer son adaptabilité à différents contextes et plus précisément aux MCSs à durée de vie courte comme les MCSs RE/Golgi, cette étude démontre également l'implication de VAP-A dans la fusion mitochondrialeVAP-A is a receptor at the surface of the endoplasmic reticulum (ER) for hundreds of proteins containing a FFAT motif and having a wide range of structures and functions. VAP-A is also required for creating multiple membrane contact sites (MCSs) between the ER and other compartments, which notably enable non-vesicular lipid exchanges between membranes. For example, the lipid-transfer protein (LTP) OSBP interacts with VAP at ER/Golgi MCS to transport cholesterol through coupled counter-exchange and hydrolysis of PI4P. It is well known that VAP-A partners contain a FFAT motif specifically recognized by the Major-Sperm-Protein (MSP) domain of VAP, however, how this receptor adapts to its different targets in MCSs that are so different in geometry and lifetime is not understood.In this study, we used a multidisciplinary approach to demonstrate that VAP-A contains two intrinsically disordered linkers that provide it with a high degree of flexibility to enable functional organization of different MCSs. A VAP-A mutant without flexible linkers is restricted in its subcellular localization, and does not support lipid transport by OSBP and CERT at ER/Golgi MCS. However, this mutant is present at ER/mitochondria MCS by interacting with VPS13A and PTPIP51, and thus facilitates lipid transport contributing to cardiolipin metabolism and mitochondrial fusion.In conclusion, this work indicates that VAP-A conformational flexibility mediated by its intrinsically disordered regions is key to ensure membrane tethering especially at short-lived MCSs; it also demonstrates the implication of VAP-A in mitochondrial fusion
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Di, Mattia Thomas. "Identification et caractérisation de la protéine MOSPD2, un bâtisseur de sites de contact membranaire impliquant le réticulum endoplasmique." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ043.
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Les sites de contact membranaire (SCM) sont des régions subcellulaires où deux organites sont physiquement connectés. Ces micro-domaines, moléculairement définis par des interactions protéine-protéine et/ou protéine-membrane, sont impliqués dans la dynamique des organites et la communication inter-organites. Le champ disciplinaire des SCM s’enrichit constamment grâce à la découverte de nouveaux acteurs moléculaires impliqués dans l'attachement des organites entre eux. Dans ce contexte de recherche, nous avons identifié un nouvel acteur impliqué dans la formation de SCM, appelé MOSPD2 (motile sperm domain-containing protein 2). Cette protéine est ancrée dans la membrane du réticulum endoplasmique (RE) ; elle peut interagir grâce à son domaine MSP avec d’autres protéines (associées à d’autres organites cellulaires) dont la caractéristique commune est de posséder un court motif protéique nommé FFAT. Par ces interactions, MOSPD2 permet l’établissement de SCM entre le RE et les endosomes, les mitochondries et l’appareil de Golgi. Ces résultats montrent une nouvelle façon de piéger des organites dans le grand filet cytoplasmique qu’est le REMembrane contact sites (MCS) are specific subcellular regions where two organelles are physically connected. Such micro-domains - molecularly defined by protein-protein and/or protein membrane interactions - are involved in organelle dynamic and inter-organelle communication. The field of MCS is constantly expanding thanks to the discovery of new molecular actors involved in organelle tethering. In this context of research, we identified MOSPD2 (motile sperm domain-containing protein 2) as a new factor involved in the formation of MCS. The MOSPD2 protein is anchored to the membrane of the endoplasmic reticulum (ER); it is able to interact thanks to its MSP domain with other organelle-associated proteins which common feature is to have a short protein motif called FFAT. By binding with its protein partners, MOSPD2 establishes MCS between the ER and endosomes, mitochondria and the Golgi apparatus. These results show how a large net covering the entire cytoplasm made by the ER can trap a large variety of cellular organelles
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Jemaiel, Aymen. "Etude du trafic membranaire vésiculaire et non-vésiculaire chez la levure." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112348/document.
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Les cellules eucaryotes sont caractérisées par le cloisonnement des organelles par des membranes. La communication entre les différents compartiments cellulaires est assurée par deux voies de transport : le transport vésiculaire et transport non-vésiculaire. Le transport vésiculaire permet à la fois le trafic des protéines et des lipides d'un compartiment à un autre, alors que le transport non-vésiculaire permet uniquement le trafic des lipides. En effet, les lipides jouent un rôle essentiel dans l'organisation cellulaire. Au cours de ma thèse, je me suis intéressé au rôle des lipides dans le trafic intracellulaire, en utilisant la levure comme organisme modèle. Dans une première partie de ma thèse, j'ai étudié les hélices amphipathiques qui permettent le ciblage des protéines vers des compartiments cellulaires spécifiques. Dans une étude précédente, réalisé au laboratoire a montré que ces hélices amphipathiques interagissaient directement avec les lipides membranaires, ce qui permet un adressage spécifique des protéines en fonction des environnements lipidiques dans la cellule. Deux hélices amphipatiques ont fait l’objet de cette étude : le motif ALPS qui cible les vésicules de la voie sécrétoire précoce, et alpha-synucléine qui reconnaît et fixe les vésicules du compartiment trans-Golgi-membrane plasmique. Dans cette première partie de la thèse j’ai cherché à identifier des motifs similaires à celui d’alpha-synucléine dans les protéines de levure, et de déterminer leurs rôles dans la cellule. Dans une seconde partie de ma thèse, en collaboration avec le laboratoire du Dr Thierry Galli, j'ai étudié de nouveaux composants impliqués dans le métabolisme lipidique aux sites de contact entre le réticulum endoplasmique et la membrane plasmique. Les sites de contact membranaires sont des régions de proches appositions (de l'ordre de 10 à 30 nm) entre deux membranes, généralement entre la membrane du réticulum endoplasmique (RE) et une autre organelle. Ce sont principalement des sites de transfert des lipides et d'ions. Maja Petkovic dans le laboratoire de Thierry Galli a fait la découverte que la protéine SNARE du RE, Sec22, interagit avec une syntaxine (Stx1) de la membrane plasmique dans les neurones, ce qui permet un nouveau mécanisme de contact entre ces deux membranes. J’ai donc essayé de voir si ce mécanisme est conservé chez la levure. Les résultats que j'ai obtenus ont confirmé que la levure Sec22 est capable d'interagir avec une protéine SNARE SSO1 localisée à la membrane plasmatique et homologue de Stx1. J'ai trouvé par co-immunoprecipitation que Sec22 et SSO1 deux interagissent avec les protéines de transfert des lipides localisées aux sites de contact. L'utilisation d'une sonde spécifique au Phosphatidylinositol-4 phosphate (PI4P), nous a permis de montrer que Sec22 est impliquée dans la régulation du niveau de PI4P à la membrane plasmique. Pour disséquer les deux fonctions de Sec22, dans la voie sécrétoire et aux sites de contact, nous avons utilisé l'approche des suppresseurs multicopies dans la levure. Parmi les suppresseurs identifiés, nous avons trouvé le Sfh1, une protéine qui a un rôle potentiel dans le transfert des lipides. Ces résultats confirment bien ceux obtenus par l’équipe de Thierry Galli, montrant que Sec22 a un nouveau rôle aux sites de contact entre le RE et la membrane plasmique et suggèrent que ce complexe SNARE pourrait être impliqué dans transfert de lipides chez la levureEukaryotic cells are characterized by their internal membrane compartmentalization, with the various specialized organelles of the cell bounded by lipid membranes. Communication between different cellular compartments occurs via two transport pathways: vesicular transport and non-vesicular transport. Vesicular transport carries both proteins and lipids from one compartment to another in cells, whereas non-vesicular transport carries only lipids. An emerging idea is the important role that lipids play in cellular organization. Lipid binding amphipathic helices such as the ALPS (amphipathic lipid packing sensor) motif are targeted to membranes of a specific lipid composition, and hence act to transfer information encoded in membrane lipids to the vesicle trafficking machinery. The lipid composition of the membranes of different organelles is therefore of great importance. One mechanism that cells use to maintain the distinct lipid compositions of organelles is lipid transport, which occurs preferentially at membrane contact sites (MCS). MCS are regions of close appositions, on the order of 10 to 30 nm, between two membranes, generally between the membrane of the endoplasmic reticulum (ER) and another organelle. In my thesis, I addressed two aspects of how lipids and their transport function in intracellular trafficking, using yeast as a model system. First, I studied amphipathic motifs that mediate targeting of proteins to specific compartments in cells. Lipid binding amphipathic helices were shown in a previous study in the laboratory to mediate specific targeting to distinct lipid environments via direct protein-lipid interactions, both in vitro and in cells. One of these, the ALPS motif, targets vesicles of the early secretory pathway. The other, alpha-synuclein, targets vesicles travelling between the late Golgi, the plasma membrane and endosomes. I studied new potential alpha-synuclein-like motifs in yeast proteins, and their roles in cells. In a second project, in collaboration with the laboratory of Dr. Thierry Galli, I studied new compenents involved in lipid metabolism at contact sites between the endoplasmic reticulum and the plasma membrane. Maja Petkovic in the laboratory of Thierry Galli made the important discovery that the ER-localized SNARE protein Sec22 interacts with a plasma membrane syntaxin in neurons, thus providing a novel mechanism for mediating close contact between these two membranes. I addressed the question of whether this mechanism is conserved in yeast. The results I obtained confirmed that yeast Sec22 is able to interact with a SNARE protein localized to the plasma membrane, Sso1. I found by co-immunoprecitation that Sec22 and Sso1 both interact with lipid transfer proteins localized to ER-plasma membrane contact sites. Using a specific probe for phosphatidylinositol-4 phosphate (PI4P), we showed that Sec22 was involved in regulating the level of PI4P at the plasma membrane. These results extend to yeast those obtained by Maja Petkovic, Thierry Galli and colleauges showing that Sec22 has a novel role at ER-plasma membrane contact sites, and suggest that this SNARE complex might be implicated in lipid transfer at these sites in yeast
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Tavassoli, Shabnam. "Endoplasmic reticulum membrane contact sites : roles in phospholipid synthesis and cell polarity." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45261.
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Barbieri, E. "CONTACT SITES BETWEEN THE ENDOPLASMIC RETICULUM AND THE PLASMA MEMBRANE CONTROL EGFR ENDOCYTOSIS." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/471217.
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The epidermal growth factor receptor (EGFR) can be internalized through different routes. While clathrin-mediated endocytosis destines EGFR for recycling and signaling, internalization through non-clathrin endocytosis (NCE) targets the receptor for degradation. Since NCE appears to be a major negative regulator of EGFR levels, a more complete picture of this pathway would likely reveal new insights into aberrant EGFR signaling observed in many types of cancer.
By combining a candidate gene approach with an unbiased proteomic approach, we have defined EGFR-NCE as molecularly distinct from other NCE pathways, relying on functional regulators not previously implicated in endocytosis. We found that reticulon 3 (RTN3), an endoplasmic reticulum (ER)-resident protein, is fundamental for NCE-mediated EGFR internalization, and that its ablation delays EGFR degradation, demonstrating that the NCE pathway is a critical regulator of the EGF-dependent cellular response. We show that, upon stimulation with high dose of EGF, RTN3 is localized in close proximity to EGFR and that it is crucial for the formation of contact sites between the ER and the plasma membrane (PM), which are needed for NCE to proceed efficiently. We also show that ER contact sites are involved in local Ca2+ release: high EGF doses induce a release of Ca2+ from the ER to the PM, which is strongly inhibited upon knockdown of RTN3. This calcium release depends on the inositol trisphosphate (IP3) cascade and is essential for the internalization of EGFR via NCE.
In conclusion, we have discovered a new clathrin-independent endocytic pathway that relies on the action of RTN3. RTN3 is necessary for the formation of contact sites between the ER and EGFR-NCE sites at the PM, which are required for IP3R-dependent local calcium release and the completion of EGFR internalization through NCE.
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Gatta, A. "Characterisation of a newly identified family of lipid transfer proteins at membrane contact sites." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1517331/.
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Non-vesicular intracellular lipid traffic is mediated by lipid transfer proteins (LTPs), which contain domains with an internal cavity that can solubilise and transfer lipids. One of the most widespread LTP folds is the Steroidogenic Acute Regulatory Transfer (StART) domain, which forms a hydrophobic pocket, and appears in proteins with different localisations and lipid specificities. The aim of this study was to characterise a new StART-like domain family, which we identified by a bioinformatics approach. I studied aspects of the localisations, functions and structural properties of six StART-like proteins in S. cerevisiae. The yeast StART-like proteins were endoplasmic reticulum (ER)-integral membrane proteins with transmembrane domains, and they localised at membrane contact sites: Lam1p/Lam3p, and Lam2p/Lam4p at junctions between ER and plasma membrane (PM); Lam5p/Lam6p at junctions between the ER and the vacuolar membrane, at nucleus-vacuole junction (NVJ) and at ER-mitochondria contacts. To study their functions, I purified the second StART-like domain of Lam4p, and I identified sterol as its lipid ligand from in vitro binding assays and in a spectroscopy approach with fluorescent ergosterol. We named the whole family LAM for Lipid transfer proteins Anchored at Membrane contact sites. The sterol binding property of the domains was related to a phenotype shared by LAM1, LAM2 and LAM3 delete strains, which showed an increased sensitivity to the sterol-sequestering polyene antifungal drug Amphotericin B (AmB). The two most sensitive strains (lam1∆ and lam3∆), displayed low sphingolipid levels, which is as yet unexplained. All AmB phenotypes were rescued by StART-like domains from the human LAMa, Lam2/4p and Lam5/6p, suggesting that these domains bind sterol. Simultaneous deletion of LAM1, LAM2, and LAM3 significantly reduced the extent of cortical ER-PM contacts, implying that they create the structure of the particularly punctate contact site they target. Finally, I started structural analysis of Lam4S2 to study the mechanism of sterol binding and to confirm our structural model.
Books on the topic "Membrane contact site"
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Tagaya, Mitsuo, and Thomas Simmen. Organelle Contact Sites: From Molecular Mechanism to Disease. Springer, 2017.
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Tagaya, Mitsuo, and Thomas Simmen. Organelle Contact Sites: From Molecular Mechanism to Disease. Springer, 2018.
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Baloh, Robert W. Breuer’s Experiments on the Semicircular Canals and Otolith Organs. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780190600129.003.0006.
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After his groundbreaking work in the mid-1860s, Josef Breuer continued to perform experiments on the inner ear balance receptors in animals. He studied the macules of fish, reptiles, and birds and noted that all these creatures had three macules arranged in the planes of the semicircular canals, perpendicular to one another. By contrast, mammals had only two macules located in the utricle (horizontal plane) and saccule (vertical plane), again perpendicular to each other. He developed the concept of “slip” to describe the movement of the otoconial membrane over the underlying sensory epithelium that occurred with linear displacement or gravity. He developed a mathematical model to hypothesize that in humans there was only one combination of responses from the two macules on each side for a single head position in space.
Book chapters on the topic "Membrane contact site"
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Pavelka, Margit, and Jürgen Roth. "Membrane Contact Sites." In Functional Ultrastructure, 194–95. Vienna: Springer Vienna, 2015. http://dx.doi.org/10.1007/978-3-7091-1830-6_12.
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Jockusch, B. M., M. Kroemker, and K. Schlüter. "Membrane-Microfilament Attachment Sites: the Art of Contact Formation." In The Cytoskeleton, 49–60. Berlin, Heidelberg: Springer Berlin Heidelberg, 1995. http://dx.doi.org/10.1007/978-3-642-79482-7_6.
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Roest, Gemma, Rita M. La Rovere, Geert Bultynck, and Jan B. Parys. "IP3 Receptor Properties and Function at Membrane Contact Sites." In Advances in Experimental Medicine and Biology, 149–78. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/978-3-319-55858-5_7.
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Derré, Isabelle. "Hijacking of Membrane Contact Sites by Intracellular Bacterial Pathogens." In Advances in Experimental Medicine and Biology, 211–23. Singapore: Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-4567-7_16.
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Harner, Max. "Isolation of Contact Sites Between Inner and Outer Mitochondrial Membranes." In Methods in Molecular Biology, 43–51. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6824-4_4.
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Tamura, Yasushi, and Toshiya Endo. "Role of Intra- and Inter-mitochondrial Membrane Contact Sites in Yeast Phospholipid Biogenesis." In Advances in Experimental Medicine and Biology, 121–33. Singapore: Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-4567-7_9.
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Del Dottore, Emanuela, Alessio Mondini, Davide Bray, and Barbara Mazzolai. "Miniature Soil Moisture Sensors for a Root-Inspired Burrowing Growing Robot." In Biomimetic and Biohybrid Systems, 184–96. Cham: Springer Nature Switzerland, 2023. http://dx.doi.org/10.1007/978-3-031-38857-6_15.
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AbstractThis paper shows the implementation of miniature sensors for soil moisture measurement and their integration in a root-inspired burrowing growing robot. Three kinds of sensors are combined to estimate the water content in soil: a resistivity sensor composed of two brass electrodes, a commercial air humidity sensor interfaced with the soil by a filter membrane of PTFE with polyester scrim, and an RGB sensor used for visible reflectance spectroscopy. We show their integration and embeddability in a burrowing growing robot based on additive manufacturing with a 4 cm probe diameter. The multimodal sensing strategy has been characterized and tested in clay and sand medium at different water content. Results show that the resistive sensor works in all the tested ranges but is prone to failure due to electrode-soil contact issues. The air humidity sensor works accurately in a range of water content less than 5% (dry conditions), and the RGB sensor works in the 5–20% range. We propose a statistical approach for soil moisture estimation that combines all three technologies and demonstrate that we can accurately predict the water content in our experimental soils, clay and sand, with better performance in clay (Root Mean Square Error, RMSE = 0.38). The proposed miniaturized multimodal sensing strategy can enable long-term, in-situ soil moisture monitoring functionalities in self-deployable robots for precision agriculture and forestry applications.
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Hanada, Kentaro. "Ceramide Transport from the Endoplasmic Reticulum to the Trans Golgi Region at Organelle Membrane Contact Sites." In Advances in Experimental Medicine and Biology, 69–81. Singapore: Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-4567-7_5.
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Enrich, Carlos, Albert Lu, Francesc Tebar, Carles Rentero, and Thomas Grewal. "Ca2+ and Annexins – Emerging Players for Sensing and Transferring Cholesterol and Phosphoinositides via Membrane Contact Sites." In Advances in Experimental Medicine and Biology, 393–438. Cham: Springer International Publishing, 2023. http://dx.doi.org/10.1007/978-3-031-21547-6_15.
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Simmen, Thomas, and Mitsuo Tagaya. "Organelle Communication at Membrane Contact Sites (MCS): From Curiosity to Center Stage in Cell Biology and Biomedical Research." In Advances in Experimental Medicine and Biology, 1–12. Singapore: Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-4567-7_1.
Full textConference papers on the topic "Membrane contact site"
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Fuentes, Daniela E., and Peter J. Butler. "Dynamics of Membrane Rafts, Talin, and Actin at Nascent and Mechanically Perturbed Focal Adhesions." In ASME 2011 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2011. http://dx.doi.org/10.1115/sbc2011-54027.
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A single endothelial cell was deformed at the apical surface by binding a functionalized nanoelectrode probe to a predetermined location on the surface of the cell. After identifying the point of contact, as recognized by the electronic signature of the nanoelectrode, and allowing binding to the cell of the fibronectin-functionalized tip, a focal adhesion site was induced at the probe site. The probe was displaced thereby applying a prescribed shear deformation to the surface of the cell. Locations of membrane rafts were identified by cholera toxin, and focal adhesion proteins were assessed using RFP-talin, and GFP-actin. Mechanical coupling and kinetics of assembly of these labeled proteins were measured using time-lapse fluorescent images taken under 60X with a multi-point confocal scanner. Raft marker GM1, Actin, and Talin were observed to sequentially accumulate at probe site with different kinetics not only upon probe contact but also upon deformation. Following deformation, later transient motion of rafts in the opposite direction of initial deformation was observed suggesting that rafts recoil. In conclusion, we report a novel nanoelectrode-based method for controlled manipulation of the cell surface and observed mechanical coupling of focal adhesions and cross-linked lipid rafts.
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Gulied, Mona, Sifani Zavahir, Tasneem Elmakki, Hazim Qiblawey, Bassim Hameed, and Dong Suk Han. "Membrane Distillation Crystallization Hybrid Process for Zero Liquid Discharge in QAFCO Plant." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2021. http://dx.doi.org/10.29117/quarfe.2021.0010.
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Qatar fertilizer company (QAFCO) is one of the world’s largest single site producer of ammonia and urea with production capacity of 12,900 metric tons per day. Currently, QAFCO faces major challenges in terms of water streams management that is generated from many processes such as wastewater from Harbor-Bosch process and brine solution from multi-stage flash (MSF) desalination process. To protect the environment; QAFCO has been making an effort to minimize the disposal of all types of water disposed into the sea. Here, this project proposes to develop a viable and economically effective process that can reach zero-liquid discharge (ZLD) of all processed water or wastewater from QAFCO facilities. The best method for ZLD is membrane distillation crystallization (MDC) hybrid process that concentrates and minimizes the volume of wastewater/brine streams to form solid through crystallizer. Membrane distillation (MD) is a thermally driven membrane process. It applies low-grade energy to create a thermal gradient across a microporous hydrophobic to vaporize water in the feed stream and condense the permeated vapor in the cold side. This research work aims to evaluate the performance of MDC for ZLD using commercial/fabricated electrospun nanofiber membrane (ENM) PVDF –base membranes at different type water streams. A general observation, higher water vapor flux and water recovery were exhibited at higher feed conductivity at 70°C. Moreover, the fabricated hydrophobic PVDF ENMs results confirmed the formation of nanofiber at the membrane surface using scanning electron microscopy (SEM). In addition, the water contact angle values of PVDF ENMs were greater than 100° and have stable mechanical and chemical properties. The ongoing research work will conduct a comparison between the optimum PVDF ENMs and the commercial MD membranes in terms of water recovery, salt rejection%, fouling/scaling, amount of collected solid and energy consumption at optimum operating conditions in MDC. In addition, it will perform a techno- economic feasibility assessment of the MDC hybrid process.
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Szatmary, Alex C., Rohan J. Banton, and Charles D. Eggleton. "Deformation of White Blood Cells Firmly Adhered to Endothelium." In ASME 2012 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/sbc2012-80894.
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Circulating white blood cells adhere to endothelium near an infection site; this occurs because infection causes ligands to be expressed on activated endothelium. Initially, a white blood cell rolls on the substrate, but eventually forms a firm adhesion, allowing it to crawl through the endothelial layer toward the infected tissue. A computational model of bond kinetics, cell deformability, and fluid dynamics was used to model the forces experienced by a cell during this process. The cell was modeled as a fluid-filled membrane; on its surface were hundreds of deformable microvilli—little fingers, ruffles in the white blood cell’s wrinkly membrane. These microvilli were deformable and their tips were decorated with PSGL-1 chemical receptors which bound to P-selectin ligands on the surface. Softer cells and cells subjected to higher fluid shear stress deformed more, and having more contact area, they formed more bonds and were able to resist more hydrodynamic load.
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Emori, Kanako, Akio Yonezu, Takumi Nagakura, and Tatsuma Miura. "Anisotropic Deformation Behavior of Porous Polymeric Membranes Under Uni-Axial and Bi-Axial Loadings." In ASME 2019 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2019. http://dx.doi.org/10.1115/imece2019-11099.
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Abstract This study systematically investigates the uniaxial and biaxial tensile deformation behavior of polytetrafluoroethylene (PTFE) membranes which are used for water purification. The present PTFE membrane has micron size pores with open cell structure, and the pore is anisotropic shape. During the uniaxial tensile test, the membranes undergo elastic deformation and plastic deformation with strain rate sensitivity (i.e. time-dependent deformation behavior). In addition, it strongly demonstrates anisotropic deformation, i.e. deformation behavior is different along longitudinal direction and transverse ones. To clarify the microscopic deformation mechanism, in-situ SEM observation is carried out during tensile loading. It is found that the anisotropic deformation behavior appears due to the inherent pore structure. Next, to investigate deformation behavior under biaxial loading condition, small punch test using a spherical indenter is carried out. The membrane undergoes elastic and plastic deformations. Finally, crack nucleates around the indenter contact and indenter completely penetrates through the membrane. It is also found that the membrane demonstrates anisotropic out-of-plane deformation behavior. To clarify these mechanisms, FEM computation is carried out, such that experimental results of force-displacement curve and out-of-plane deformation behavior are compared with the computational ones. The present FEM model enables the prediction of the membrane’s deformation behavior under biaxial loading.
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Ozolina, N. V. "MEMBRANE CONTACT SITES TO PROTECT CELLS FROM STRESS." In The All-Russian Scientific Conference with International Participation and Schools of Young Scientists "Mechanisms of resistance of plants and microorganisms to unfavorable environmental". SIPPB SB RAS, 2018. http://dx.doi.org/10.31255/978-5-94797-319-8-580-583.
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Ozolina, N. V. "THE ROLE MEMBRANE CONTACT SITES IN CELL LIFE." In The Second All-Russian Scientific Conference with international participation "Regulation Mechanisms of Eukariotic Cell Organelle Functions". SIPPB SB RAS, 2018. http://dx.doi.org/10.31255/978-5-94797-318-1-77-79.
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Vera Alvarado, Elmmer A., Md Abdur Rahman Bin Abdus Salam, Ali Ashraf, and Karen Lozano. "Graphene Reinforced PVDF Nanofibers Fabricated With the ForceSpinning® Method for Water Desalination Applications." In ASME 2023 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2023. http://dx.doi.org/10.1115/imece2023-113900.
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Abstract Direct contact membrane distillation (DCMD) is a thermally driven energy and cost-efficient desalination technique where low-grade energy from solar or waste heat from plants can be used to recover fresh water from high saline water. The DCMD technique allows both the feed and permeate solutions to be in contact with the membrane, where vapor from the feed gets condensed in the permeate side once it collides with low-temperature water. Polyvinylidene fluoride (PVDF) nanofiber membranes were prepared by the ForceSpinning® method, these were later dip coated in graphene solutions prepared with different solvents. The ForceSpinning® method, unlike electrospinning, operates in the absence of electromagnetic fields, solely based on the use of centrifugal spinning where nanofiber production is mainly controlled by the angular velocity applied to the spinneret containing the polymer solution or melt. A 0.75 wt.% graphene solution showed adequate dispersion and distribution on the developed PVDF nanofiber membrane as confirmed by Scanning Electron Microscopy (SEM). Water contact angle and surface roughness varied depending on the percentages of graphene in the coating solution. Testing parameters such as temperature, flow rate, porosity etc. were kept constant. Stainless steel filtering wire cloth of 165*1400 mesh size with 6% opening was used to support the PVDF membrane. Among the registered fluxes after testing, 12.82 Kg m−2hr−1 and 10.98 Kg m−2hr−1 were the most relevant. To reduce the clogging during the experimentation, membranes with an EcoFlex support were additionally tested. This study presents a promising platform for moderate to high salinity rejection, as well as produced water desalination.
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Oh, Kyung Su, Seungho Park, Ohmyoung Kwon, Young Ki Choi, and Joon Sik Lee. "Molecular Dynamics Simulation of Water Behavior as a Function of Temperatures and Monomer Numbers in Nafion 117." In 2007 First International Conference on Integration and Commercialization of Micro and Nanosystems. ASMEDC, 2007. http://dx.doi.org/10.1115/mnc2007-21413.
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The proton exchange membrane plays a critical role as an electrolyte for proton transports in the PEMFC. Generally, the membrane, such as Nafion 117, consists of a polytetrafluoro-ethylene (PTFE) backbone and side-chains terminated with a sulfonate group (SO3−). Operating the fuel cell, the membrane preferentially becomes hydrated by absorbing water. Then, the hydrogen atom on the SO3− part of the side-chain can detach from its own position and hop to the next SO3− site. The water management is the key to the efficient operation of the fuel cell, since the water content is the one of decisive factors for membrane’s lifetime and efficient operations of fuel cells as well. In this report, we set up the molecular model for hydrated Nafion 117 and simulate the molecular movements for various temperatures and monomer numbers. Here, we obtain the mean square displacements of water molecules and estimate the self-diffusion coefficients of water in the Nafion 117.
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Creasy, M. Austin, and Donald J. Leo. "Non-Invasive Measurement Techniques for Measuring Bilayers in Droplet-Interface-Bilayers." In ASME 2009 Conference on Smart Materials, Adaptive Structures and Intelligent Systems. ASMEDC, 2009. http://dx.doi.org/10.1115/smasis2009-1321.
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Phospholipids and membrane proteins are two of the fundamental building blocks of cell membranes in living organisms. These molecules are amphipathic and synthetic membranes made of phospholipids, called bilayer lipid membranes (BLMs), are used to understand the characteristics of a cell membrane. Studies of these BLMs have been performed on both solid support and liquid support systems. A droplet interface bilayer (DIB) is a liquid support system where a monolayer is formed around a water droplet placed in oil and a bilayer is formed when two of these droplets are placed in contact. For impedance measurements, electrodes are placed in the water on each side of the bilayer. The measurements first insure there is a bilayer and second to obtain information about the bilayer. In a DIB system the electrodes pierce the monolayer surrounding the droplet causing instabilities in the monolayer. This study focuses on a non-invasive technique for measuring the bilayer by using the electrodes to contact the monolayer around the droplets and take impedance measurements without piercing the monolayer.
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Limaye, Mukta S., and James F. Klausner. "Performance of a Flexible Evaporator for Loop Heat Pipe Applications." In ASME 2008 Heat Transfer Summer Conference collocated with the Fluids Engineering, Energy Sustainability, and 3rd Energy Nanotechnology Conferences. ASMEDC, 2008. http://dx.doi.org/10.1115/ht2008-56128.
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A flat and flexible evaporator, which conforms to contoured surfaces, has been developed for loop heat pipe applications. A loop heat pipe (LHP) is a passive, two phase heat transfer device that uses a porous membrane in the evaporator to circulate fluid. A number of flexible membranes have been tested as evaporator wicks that have a length of 12.7 cm and heated area of 50.6 cm2. For cellulose, polyethylene, and blotting paper membranes, maximum heat fluxes of 0.43, 1.5 and 2.9 W/cm2 have been observed, respectively. The maximum heat transfer coefficients measured for these membranes are 551, 876, and 2100 W/m2-K, respectively. The best performance was observed by a membrane made of a fibrous cotton matrix, typically used as gauze. This material has a large pore size and high wettability with water. When tested in a rigid, brass evaporator, the maximum heat flux observed is 5.95 W/cm2, and the maximum heat transfer coefficient is 2865 W/m2-K. A flexible evaporator is fabricated using a heat sealable, flexible barrier pouch, and the cotton matrix membrane is sealed inside. The maximum measured heat flux for the flexible evaporator is 3.2 W/cm2 and maximum measured heat transfer coefficient is 1165 W/m2-K. The observed reduction in heat transfer as compared to the rigid evaporator is due to the poor contact between the evaporator and membrane. It is concluded that for the flexible evaporator membranes considered, the heat transfer mechanism is boiling and the maximum heat flux is limited by the wicking rate of the membrane. For a given membrane, the wicking rate increases with a reduction in the wicking length and decreases with an increasing rate of evaporation. To further improve the performance of the flexible evaporator, it is necessary to ensure efficient vapor removal from the evaporator as well as maintaining good contact between the membrane and the evaporator surface.
Reports on the topic "Membrane contact site"
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Warrick, Arthur, Uri Shani, Dani Or, and Muluneh Yitayew. In situ Evaluation of Unsaturated Hydraulic Properties Using Subsurface Points. United States Department of Agriculture, October 1999. http://dx.doi.org/10.32747/1999.7570566.bard.
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The primary information for accurately predicting water and solute movement and their impact on water quality is the characterization of soil hydraulic properties. This project was designed to develop methods for rapid and reliable estimates of unsaturated hydraulic properties of the soil. Particularly, in situ methodology is put forth, based on subsurface point sources. Devices were designed to allow introduction of water in subsurface settings at constant negative heads. The ability to operate at a negative head allows a direct method of finding unsaturated soil properties and a mechanism for eliminating extremely rapid preferential flow from the slow matrix flow. The project included field, laboratory and modeling components. By coupling the measurements and the modeling together, a wider range of designs can be examined, while at the same time realistic performance is assured. The developed methodology greatly expands the possibilities for evaluating hydraulic properties in place, especially for measurements in undisturbed soil within plant rooting zones. The objectives of the project were (i) To develop methods for obtaining rapid and reliable estimates of unsaturated hydraulic properties in situ, based on water distribution from subsurface point sources. These can be operated with a constant flow or at a constant head; (ii) To develop methods for distinguishing between matrix and preferential flow using cavities/permeameters under tension; (iii) To evaluate auxiliary measurements such as soil water content or tensions near the operating cavities to improve reliability of results; and (iv: To develop numerical and analytical models for obtaining soil hydraulic properties based on measurements from buried-cavity sources and the auxiliary measurements. The project began in July 1995 and was terminated in November 1998. All of the objectives were pursued. Three new subsurface point sources were designed and tested and two old types were also used. Two of the three new designs used a nylon cloth membrane (30 mm) arranged in a cylindrical geometry and operating at a negative water pressure (tension). A separate bladder arrangement allowed inflation under a positive pressure to maintain contact between the membrane and the soil cavity. The third new design used porous stainless steel (0.5 and 5 mm) arranged in six segments, each with its own water inlet, assembled to form a cylindrical supply surface when inflated in a borehole. The "old" types included an "off-the-shelf" porous cup as well as measurements from a subsurface drip emitter in a small subsurface cavity. Reasonable measurements were made with all systems. Sustained use of the cloth membrane devices were difficult because of leaks and plugging problems. All of the devices require careful consideration to assure contact with the soil system. Steady flow was established which simplified the analysis (except for the drip emitter which used a transient analysis).
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Wisniewski, Michael, Samir Droby, John Norelli, Dov Prusky, and Vera Hershkovitz. Genetic and transcriptomic analysis of postharvest decay resistance in Malus sieversii and the identification of pathogenicity effectors in Penicillium expansum. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597928.bard.
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Use of Lqh2 mutants (produced at TAU) and rNav1.2a mutants (produced at the US side) for identifying receptor site-3: Based on the fact that binding of scorpion alpha-toxins is voltage-dependent, which suggests toxin binding at the mobile voltage-sensing region, we analyzed which of the toxin bioactive domains (Core-domain or NC-domain) interacts with the DIV Gating-module of rNav1.2a. This analysis was based on the assumption that the dissociation of toxin mutants upon depolarization would vary from that of the unmodified toxin should the substitutions affect a site of interaction with the channel Gating-module. Using a series of toxin mutants (mutations at both domains) and two channel mutants that were shown to reduce the sensitivity to scorpion alpha-toxins, and by comparison of depolarization-driven dissociation of Lqh2 derivatives off their binding site at rNav1.2a mutant channels we found that the toxin Core-domain interacts with the Gating-module of DIV. Details of the experiments and results appear in Guret al (2011). Mapping receptor site 3 at Nav1.2a by extensive channel mutagenesis (Seattle): Since previous studies with photoaffinity labeling and antibody mapping implicated domains I and IV in scorpion alpha-toxin binding, Nav1.2 channel mutants containing substitutions at these extracellular regions were expressed and tested for receptor function by whole-cell voltage clamp. Of a large number of channel mutants, T1560A, F1610A, and E1613A in domain IV had ~5.9-, ~10.7-, and ~3.9-fold lower affinities for the scorpion toxin Lqh2, respectively, and mutant E1613R had 73-fold lower affinity. Toxin dissociation was accelerated by depolarization for both wild-type and mutants, and the rates of dissociation were also increased by mutations T1560A, F1610A and E1613A. In contrast, association rates for these three mutant channels at negative membrane potentials were not significantly changed and were not voltage-dependent. These results indicated that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also showed a ~3.4-fold lower affinity for Lqh2, indicating that this extracellular loop may form a secondary component of the toxin binding site. Analysis with the Rosetta-Membrane algorithm revealed a three-dimensional model of Lqh2 binding to the voltage sensor in a resting state. In this model, amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV that are important for toxin binding interact with amino acid residues on two faces of the wedge-shaped Lqh2 molecule that are important for toxin action. The conserved gating charges in the S4 transmembrane segment are in an inward position and likely form ion pairs with negatively charged amino acid residues in the S2 and S3 segments (Wang et al 2011; Gurevitz 2012; Gurevitzet al 2013).
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Upadhyaya, Shrini K., Abraham Shaviv, Abraham Katzir, Itzhak Shmulevich, and David S. Slaughter. Development of A Real-Time, In-Situ Nitrate Sensor. United States Department of Agriculture, March 2002. http://dx.doi.org/10.32747/2002.7586537.bard.
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Although nitrate fertilizers are critical for enhancing crop production, excess application of nitrate fertilizer can result in ground water contamination leading to the so called "nitrate problem". Health and environmental problems related to this "nitrate problem" have led to serious concerns in many parts of the world including the United States and Israel. These concerns have resulted in legislation limiting the amount of nitrate N in drinking water to 10mg/g. Development of a fast, reliable, nitrate sensor for in-situ application can be extremely useful in dynamic monitoring of environmentally sensitive locations and applying site-specific amounts of nitrate fertilizer in a precision farming system. The long range objective of this study is to develop a fast, reliable, real-time nitrate sensor. The specific objective of this one year feasibility study was to explore the possible use of nitrate sensor based on mid-IR spectroscopy developed at UCD along with the silver halide fiber ATR (i.e. attenuated total internal reflection) sensor developed at TAU to detect nitrate content in solution and soil paste in the presence of interfering compounds. Experiments conducted at Technion and UCD clearly demonstrate the feasibility of detecting nitrate content in solutions as well as soil pastes using mid-IR spectroscopy and an ATR technique. When interfering compounds such as carbonates, bicarbonates, organic matter etc. are present special data analysis technique such as singular value decomposition (SYD) or cross correlation was necessary to detect nitrate concentrations successfully. Experiments conducted in Israel show that silver halide ATR fiber based FEWS, particularly flat FEWS, resulted in low standard error and high coefficient of determination (i.e. R² values) indicating the potential of the flat Fiberoptic Evanescent Wave Spectroscopy (FEWS) for direct determinations of nitrate. Moreover, they found that it was possible to detect nitrate and other anion concentrations using anion exchange membranes and M1R spectroscopy. The combination of the ion-exchange membranes with fiberoptices offers one more option to direct determination of nitrate in environmental systems.
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Adam, Zach, and Eran Pichersky. Degradation of Abnormal Proteins in Chloroplasts of Higher Plants. United States Department of Agriculture, August 1994. http://dx.doi.org/10.32747/1994.7568768.bard.
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In this study we attempted to get a better understanding of processes involved in the degradation of abnormal proteins i chloroplasts. To achieve this goal, we used a number of complementary approaches. We first characterized the expression of the two subunits of Clp protease. We demonstrated that both of them were expressed in chloroplasts in a constitutive fashion, but the expression of the regulatory subunit ClpC was enhanced by light. We generated a mutant the lumenal protein OEE33 which was targeted to the stroma in in vitro experiments. In the wrong compartment it was found unstable, and characterization of its degradation revealed that it was degraded by a soluble, ATP-dependent serine protease, which are also the characteristics of Clp protease. In search of other homologues of bacterial proteases, we found that chloroplasts contain a homologue of the FtsH protease. It is an ATP-dependent metallo-protease, bound to the stromal side of the thylakoid membrane, whose expression is dependent on light. The gene encodig this protease was cloned and characterized. In attempt to generate Arabidopsis mutant plants impaired in their capability to degrade abnormal chloroplast proteins, we fused the gene for mistargeted OEE33 to the streptomycin-detoxifying gene. This chimeric gene was introduced into Arabodipsis plants, to generate transformed plants. This transformants plants were sensitive to streptomycin due to the rapid turn-over of the chimeric protein. Seeds from these plants were then chemically mutagenised, and seedlings were selected for their capability to grow on streptomycin. The ability of these mutant transformants to grow on streptomycin is presumably due to stabilization of the chimeric protein. These plants will allow us in the future to identify the effected genes, which are likely to be involved in the protein degradation process.
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Delwiche, Michael, Boaz Zion, Robert BonDurant, Judith Rishpon, Ephraim Maltz, and Miriam Rosenberg. Biosensors for On-Line Measurement of Reproductive Hormones and Milk Proteins to Improve Dairy Herd Management. United States Department of Agriculture, February 2001. http://dx.doi.org/10.32747/2001.7573998.bard.
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The original objectives of this research project were to: (1) develop immunoassays, photometric sensors, and electrochemical sensors for real-time measurement of progesterone and estradiol in milk, (2) develop biosensors for measurement of caseins in milk, and (3) integrate and adapt these sensor technologies to create an automated electronic sensing system for operation in dairy parlors during milking. The overall direction of research was not changed, although the work was expanded to include other milk components such as urea and lactose. A second generation biosensor for on-line measurement of bovine progesterone was designed and tested. Anti-progesterone antibody was coated on small disks of nitrocellulose membrane, which were inserted in the reaction chamber prior to testing, and a real-time assay was developed. The biosensor was designed using micropumps and valves under computer control, and assayed fluid volumes on the order of 1 ml. An automated sampler was designed to draw a test volume of milk from the long milk tube using a 4-way pinch valve. The system could execute a measurement cycle in about 10 min. Progesterone could be measured at concentrations low enough to distinguish luteal-phase from follicular-phase cows. The potential of the sensor to detect actual ovulatory events was compared with standard methods of estrus detection, including human observation and an activity monitor. The biosensor correctly identified all ovulatory events during its testperiod, but the variability at low progesterone concentrations triggered some false positives. Direct on-line measurement and intelligent interpretation of reproductive hormone profiles offers the potential for substantial improvement in reproductive management. A simple potentiometric method for measurement of milk protein was developed and tested. The method was based on the fact that proteins bind iodine. When proteins are added to a solution of the redox couple iodine/iodide (I-I2), the concentration of free iodine is changed and, as a consequence, the potential between two electrodes immersed in the solution is changed. The method worked well with analytical casein solutions and accurately measured concentrations of analytical caseins added to fresh milk. When tested with actual milk samples, the correlation between the sensor readings and the reference lab results (of both total proteins and casein content) was inferior to that of analytical casein. A number of different technologies were explored for the analysis of milk urea, and a manometric technique was selected for the final design. In the new sensor, urea in the sample was hydrolyzed to ammonium and carbonate by the enzyme urease, and subsequent shaking of the sample with citric acid in a sealed cell allowed urea to be estimated as a change in partial pressure of carbon dioxide. The pressure change in the cell was measured with a miniature piezoresistive pressure sensor, and effects of background dissolved gases and vapor pressures were corrected for by repeating the measurement of pressure developed in the sample without the addition of urease. Results were accurate in the physiological range of milk, the assay was faster than the typical milking period, and no toxic reagents were required. A sampling device was designed and built to passively draw milk from the long milk tube in the parlor. An electrochemical sensor for lactose was developed starting with a three-cascaded-enzyme sensor, evolving into two enzymes and CO2[Fe (CN)6] as a mediator, and then into a microflow injection system using poly-osmium modified screen-printed electrodes. The sensor was designed to serve multiple milking positions, using a manifold valve, a sampling valve, and two pumps. Disposable screen-printed electrodes with enzymatic membranes were used. The sensor was optimized for electrode coating components, flow rate, pH, and sample size, and the results correlated well (r2= 0.967) with known lactose concentrations.
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Ohad, Itzhak, and Himadri Pakrasi. Role of Cytochrome B559 in Photoinhibition. United States Department of Agriculture, December 1995. http://dx.doi.org/10.32747/1995.7613031.bard.
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The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.
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Dubcovsky, Jorge, Tzion Fahima, and Ann Blechl. Molecular characterization and deployment of the high-temperature adult plant stripe rust resistance gene Yr36 from wheat. United States Department of Agriculture, November 2013. http://dx.doi.org/10.32747/2013.7699860.bard.
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Stripe rust, caused by Puccinia striiformis f. sp. tritici is one of the most destructive fungal diseases of wheat. Virulent races that appeared within the last decade caused drastic cuts in yields. The incorporation of genetic resistance against this pathogen is the most cost-effective and environmentally friendly solution to this problem. However, race specific seedling resistance genes provide only a temporary solution because fungal populations rapidly evolve to overcome this type of resistance. In contrast, high temperature adult plant (HTAP) resistance genes provide a broad spectrum resistance that is partial and more durable. The cloning of the first wheat HTAP stripe rust resistance gene Yr36 (Science 2009, 323:1357), funded by our previous (2007-2010) BARD grant, provided us for the first time with an entry point for understanding the mechanism of broad spectrum resistance. Two paralogous copies of this gene are tightly linked at the Yr36 locus (WKS1 and WKS2). The main objectives of the current study were to characterize the Yr36 (WKS) resistance mechanism and to identify and characterize alternative WKSgenes in wheat and wild relatives. We report here that the protein coded by Yr36, designated WKS1, that has a novel architecture with a functional kinase and a lipid binding START domain, is localized to chloroplast. Our results suggest that the presence of the START domain may affect the kinase activity. We have found that the WKS1 was over-expressed on leaf necrosis in wheat transgenic plants. When the isolated WKS1.1 splice variant transcript was transformed into susceptible wheat it conferred resistance to stripe rust, but the truncated variant WKS1.2 did not confer resistance. WKS1.1 and WKS1.2 showed different lipid binding profiling. WKS1.1 enters the chloroplast membrane, while WKS1.2 is only attached outside of the chloroplast membrane. The ascorbate peroxidase (APX) activity of the recombinant protein of TmtAPXwas found to be reduced by WKS1.1 protein in vitro. The WKS1.1 mature protein in the chloroplast is able to phosphorylate TmtAPXprotein in vivo. WKS1.1 induced cell death by suppressing APX activity and reducing the ability of the cell to detoxify reactive oxygen. The decrease of APX activity reduces the ability of the plant to detoxify the reactive H2O2 and is the possible mechanism underlying the accelerated cell death observed in the transgenic plants overexpressing WKS1.1 and in the regions surrounding a stripe rust infection in the wheat plants carrying the natural WKS1.1 gene. WKS2 is a nonfunctional paralog of WKS1 in wild emmer wheat, probably due to a retrotransposon insertion close to the alternative splicing site. In some other wild relatives of wheat, such as Aegilops comosa, there is only one copy of this gene, highly similar to WKS2, which is lucking the retrotransposon insertion. WKS2 gene present in wheat and WKS2-Ae from A. showed a different pattern of alternative splice variants, regardless of the presence of the retrotransposon insertion. Susceptible Bobwhite transformed with WKS2-Ae (without retrotansposon insertion in intron10), which derived from Aegilops comosaconferred resistance to stripe rust in wheat. The expression of WKS2-Ae in transgenic plants is up-regulated by temperature and pathogen infection. Combination of WKS1 and WKS2-Ae shows improved stripe rust resistance in WKS1×WKS2-Ae F1 hybrid plants. The obtained results show that WKS1 protein is accelerating programmed cell death observed in the regions surrounding a stripe rust infection in the wheat plants carrying the natural or transgenic WKS1 gene. Furthermore, characterization of the epistatic interactions of Yr36 and Yr18 demonstrated that these two genes have additive effects and can therefore be combined to increase partial resistance to this devastating pathogen of wheat. These achievements may have a broad impact on wheat breeding efforts attempting to protect wheat yields against one of the most devastating wheat pathogen.
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Hochman, Ayala, Thomas Nash III, and Pamela Padgett. Physiological and Biochemical Characterization of the Effects of Oxidant Air Pollutants, Ozone and Gas-phase Nitric Acid, on Plants and Lichens for their Use as Early Warning Biomonitors of these Air Pollutants. United States Department of Agriculture, January 2011. http://dx.doi.org/10.32747/2011.7697115.bard.
Full textAbstract:
Introduction. Ozone and related oxidants are regarded as the most important phytotoxic air pollutant in many parts of the western world. A previously unrecognized component of smog, nitric acid, may have even greater deleterious effects on plants either by itself or by augmenting ozone injury. The effects of ozone on plants are well characterized with respect to structural and physiological changes, but very little is known about the biochemical changes in plants and lichens exposed to ozone and/or HNO3. Objectives.To compare and contrast the responses of crop plants and lichens to dry deposition of HNO3 and O3., separately, and combined in order to assess our working hypothesis that lichens respond to air pollution faster than plants. Lichens are most suitable for use as biomonitors because they offer a live-organism-based system that does not require maintenance and can be attached to any site, without the need for man-made technical support systems. Original Immediate aims To expose the tobacco (Nicotiana tabacum L.) cultivar Bel-W3 that is ozone supersensitive and the ozone sensitive red kidney bean (Phaseolusvulgaris) and the lichen Ramalinamenziesii to controlled HNO3 and O3 fumigations and combined and to follow the resulting structural, physiological and biochemical changes, with special reference to reactive oxygen species related parameters. Revised. Due to technical problems and time limitations we studied the lichen Ramalinamenziesii and two cultivar of tobacco: Bel-W3 that is ozone supersensitive and a resistant cultivar, which were exposed to HNO3 and O3 alone (not combined). Methodology. Plants and lichens were exposed in fumigation experiments to HNO3 and O3, in constantly stirred tank reactors and the resulting structural, physiological and biochemical changes were analyzed. Results. Lichens. Exposure of Ramalinamenziesiito HNO3 resulted in cell membrane damage that was evident by 14 days and continues to worsen by 28 days. Chlorophyll, photosynthesis and respiration all declined significantly in HNO3 treatments, with the toxic effects increasing with dosage. In contrast, O3 fumigations of R. menziesii showed no significant negative effects with no differences in the above response variables between high, moderate and low levels of fumigations. There was a gradual decrease in catalase activity with increased levels of HNO3. The activity of glutathione reductase dropped to 20% in thalli exposed to low HNO3 but increased with its increase. Glucose 6-phosphate dehydrogenase activity increase by 20% with low levels of the pollutants but decreased with its increase. Tobacco. After 3 weeks of exposure of the sensitive tobacco cultivar to ozone there were visible symptoms of toxicity, but no danmage was evident in the tolerant cultivar. Neither cultivar showed any visible symptoms after exposure to HNO3.In tobacco fumigated with O3, there was a significant decrease in maximum photosynthetic CO2 assimilation and stomatal conductance at high levels of the pollutant, while changes in mesophyll conductance were not significant. However, under HNO3 fumigation there was a significant increase in mesophyll conductance at low and high HNO3 levels while changes in maximum photosynthetic CO2 assimilation and stomatal conductance were not significant. We could not detect any activity of the antioxidant enzymes in the fumigated tobacco leaves. This is in spite of the fact that we were able to assay the enzymes in tobacco leaves grown in Israel. Conclusions. This project generated novel data, and potentially applicable to agriculture, on the differential response of lichens and tobacco to HNO3 and O3 pollutants. However, due to experimental problems and time limitation discussed in the body of the report, our data do not justify yet application for a full, 4-year grant. We hope that in the future we shall conduct more experiments related to our objectives, which will serve as a basis for a larger scale project to explore the possibility of using lichens and/or plants for biomonitoring of ozone and nitric acid air pollution.
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Steffens, John C., and Eithan Harel. Polyphenol Oxidases- Expression, Assembly and Function. United States Department of Agriculture, January 1995. http://dx.doi.org/10.32747/1995.7571358.bard.
Full textAbstract:
Polyphenol oxidases (PPOs) participate in the preparation of many plant products on the one hand and cause considerable losses during processing of plant products on the other hand. However, the physiological functions of plant PPO were still a subject of controversy at the onset of the project. Preliminary observations that suggested involvement of PPOs in resistance to herbivores and pathogens held great promise for application in agriculture but required elucidation of PPO's function if modulation of PPO expression is to be considered for improving plant protection or storage and processing of plant products. Suggestions on a possible role of PPO in various aspects of chloroplast metabolism were also relevant in this context. The characterization of plant PPO genes opened a way for achieving these goals. We reasoned that "understanding PPO targeting and routing, designing ways to manipulate its expression and assessing the effects of such modifications will enable determination of the true properties of the enzyme and open the way for controlling its activity". The objective of the project was to "obtain an insight into the function and biological significance of PPOs" by examining possible function(s) of PPO in photosynthesis and plant-pest interactions using transgenic tomato plants; extending our understanding of PPO routing and assembly and the mechanism of its thylakoid translocation; preparing recombinant PPOs for use in import studies, determination of the genuine properties of PPOs and understanding its assembly and determining the effect of PPO's absence on chloroplast performance. Results obtained during work on the project made it necessary to abandon some minor objectives and devote the effort to more promising topics. Such changes are mentioned in the 'Body of the report' which is arranged according to the objectives of the original proposal. The complex expression pattern of tomato PPO gene family was determined. Individual members of the family are differentially expressed in various parts of the plant and subjected to developmentally regulated turnover. Some members are differentially regulated also by pathogens, wounding and chemical wound signals. Wounding systemically induces PPO activity and level in potato. Only tissues that are developmentally competent to express PPO are capable of responding to the systemic wounding signal by increased accumulation of PPO mRNA. Down regulation of PPO genes causes hyper susceptibility to leaf pathogens in tomato while over expression regulation of PPO expression in tomato plants is their apparent increased tolerance to drought. Both the enhanced disease resistance conferred by PPO over expression and the increased stress tolerance due to down regulation can be used in the engineering of improved crop plants. Photosynthesis rate and variable fluorescence measurements in wild type, and PPO-null and over expressing transgenic tomato lines suggest that PPO does not enable plants to cope better with stressful high light intensities or reactive oxygen species. Rather high levels of the enzyme aggravate the damage caused under such conditions. Our work suggests that PPO's primary role is in defending plants against pathogens and herbivores. Jasmonate and ethylene, and apparently also salicylate, signals involved in responses to wounding and defense against herbivores and pathogens, enhance markedly and specifically the competence of chloroplasts to import and process pPPO. The interaction of the precursor with thylakoid membranes is primarily affected. The routing of PPO shows other unusual properties: stromal processing occurs in two sites, resulting in intermediates that are translocated across thylakoids by two different mechanisms - a DpH- and a Sec-dependent one. It is suggested that the dual pattern of processing and routing constitutes a'fail safe' mechanism, reflecting the need for a rapid and flexible response to defense challenges. Many of the observations described above should be taken into consideration when manipulation of PPO expression is contemplated for use in crop improvement.