The Experts below are selected from a list of 132 Experts worldwide ranked by ideXlab platform
Roy Duncan - One of the best experts on this subject based on the ideXlab platform.
-
the p14 fusion associated small transmembrane fast protein effects membrane fusion from a subset of membrane microdomains
Journal of Biological Chemistry, 2006Co-Authors: Jennifer A Corcoran, Jayme Salsman, Roberto De Antueno, Ahmed Touhami, M H Jericho, Eileen K Clancy, Roy DuncanAbstract:Abstract The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.
-
the p14 fusion associated small transmembrane fast protein effects membrane fusion from a subset of
2006Co-Authors: Jennifer A Corcoran, Jayme Salsman, Roberto De Antueno, Ahmed Touhami, M H Jericho, Eileen K Clancy, Roy Duncan, Fromthedepartmentsof MicrobiologyandimmunologyandAbstract:The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WXand Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.
Jean-louis Delaunay - One of the best experts on this subject based on the ideXlab platform.
-
1 Differential solubilization of inner plasma membrane leaflet components by
2009Co-Authors: Triton X, Jean-louis Delaunay, Germain Trugnan, Michèle Maurice, Michelyne Breton, Inserm U, Faculté De Médecine Saint-antoineAbstract:A commonly-used method for analyzing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4°C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and inserm-00418165, version 1- 17 Sep 2009 Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.3 1
-
Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100.
Biochimica et Biophysica Acta:Biomembranes, 2008Co-Authors: Jean-louis Delaunay, Breton Michelyne, Germain Trugnan, Michèle MauriceAbstract:A commonly-used method for analyzing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4°C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.
-
Rôle des microdomaines membranaires dans le ciblage apical de la nucléotide pyrophosphatase NPP3 dans les cellules MDCK
2007Co-Authors: Jean-louis DelaunayAbstract:La membrane plasmique des cellules épithéliales polarisées comporte deux domaines distincts, le domaine apical et le domaine basolatéral. Chaque domaine a une composition en lipides et en protéines déterminée, leur permettant d'assurer des fonctions spécifiques. Les mécanismes moléculaires responsables du tri et de l'adressage des protéines transmembranaires vers le pôle apical sont encore mal connus. La membrane apicale est enrichie en glycosphingolipides et en cholestérol qui forment des microdomaines appelés « rafts ». Expérimentalement, les rafts peuvent être isolés sous forme de DRM (detergent-resistant membranes) définis par leur résistance à un détergent non ionique, le Triton X-100. Il a été proposé que les rafts recrutent les protéines apicales au niveau du réseau trans-golgien et servent de plateforme pour leur adressage au pôle apical. Effectivement les protéines ancrées par le glycosylphosphatidyl-inositol sont résistantes au Triton et sont localisées en général à la membrane apicale. En revanche, la plupart des protéines transmembranaires apicales sont solubles dans le Triton, bien qu'elles soient résistantes à l'action de détergents plus doux comme le Lubrol WX. L'objectif des travaux de thèse a été d'étudier le rôle des rafts dans l'adressage apical de protéines transmembranaires et de comprendre l'effet différentiel du Triton et du Lubrol sur leur solubilisation. Les nucléotides pyrophosphatases NPP1 (basolatérale) et NPP3 (apicale) exprimées de façon stable dans les cellules MDCK ont servi de modèles. NPP3 est insoluble dans le Lubrol et partiellement insoluble dans le Triton, tandis que NPP1 est essentiellement solubilisée. L'étude de la localisation et de la sensibilité aux détergents de mutants et de chimères combinant des domaines cytoplasmiques, transmembranaires et extracellulaires de NPP3 et NPP1, a montré qu'il n'existait pas de corrélation stricte entre l'adressage apical et la résistance aux détergents. La résistance de NPP3 à la solubilisation par le Lubrol est acquise précocement au cours de sa biosynthèse, indépendamment de sa destination finale. Cette résistance dépend d'acides aminés chargés positivement situés dans la queue cytoplasmique, proches de la membrane. Afin de comprendre la sélectivité du Triton et du Lubrol dans l'extraction des protéines et des lipides membranaires, la composition lipidique des DRM obtenus après extraction par le Triton et le Lubrol a été comparée. Les DRM extraits par le Triton et le Lubrol sont enrichis en cholestérol ce qui correspond à la définition des rafts. Cependant, les DRM Triton sont appauvris en lipides du feuillet interne tandis que les DRM Lubrol sont enrichis en phosphatidyléthanolamine. Les DRM Lubrol sont également enrichis en protéines associées au feuillet interne de la membrane. En conclusion, ces travaux montrent que la résistance de la protéine apicale NPP3 à l'extraction par le Lubrol, et en partie par le Triton, est une propriété intrinsèque qui correspond probablement à une adaptation de la protéine à la composition lipidique du domaine apical, mais que cette propriété ne détermine pas son adressage polarisé. De plus, ces travaux montrent que les détergents sont des outils très intéressants pour étudier les interactions entre les protéines et les lipides membranaires, mais qu'il n'existe probablement pas de détergent capable d'isoler de façon stricte des microdomaines membranaires tels que sont définis les rafts. Nos résultats suggèrent que le feuillet interne des rafts est enrichi en phosphatidyléthanolamine et en cholestérol, qu'il est en partie solubilisé par le Triton, ce qui déstabiliserait les protéines transmembranaires et entraînerait leur extraction. Mots clés: détergent, raft, , ciblage apical, cholestérol, microdomaine membranaire, feuillet interne de la membrane, phosphatidyléthanolamine.
-
Differential detergent resistance of the apical and basolateral NPPases: relationship with polarized targeting.
Journal of Cell Science, 2007Co-Authors: Jean-louis Delaunay, Germain Trugnan, Michelyne Breton, James Goding, Michèle MauriceAbstract:Targeting of glycosylphosphatidylinositol-anchored proteins to the apical surface of epithelial cells involves clustering in Triton X-100-resistant membrane microdomains or rafts. The role of these microdomains in sorting transmembrane proteins is more questionable because, unlike glycosylphosphatidylinositol-anchored proteins, apical transmembrane proteins are rather soluble in Triton X-100. They are, however, resistant to milder detergents such as Lubrol WX or Tween 20. It has been proposed that specific membrane microdomains, defined by resistance to these detergents, would carry transmembrane proteins to the apical surface. We have used MDCK cells stably transfected with the apical and basolateral pyrophosphatases/phosphodiesterases, NPP3 and NPP1, to examine the relationship between detergent resistance and apical targeting. The apically expressed wild-type NPP3 was insoluble in Lubrol WX whereas wild-type NPP1, which is expressed basolaterally, was essentially soluble. By using tail mutants and chimeric constructs that combine the cytoplasmic, transmembrane and extracellular domains of NPP1 and NPP3, we show that there is not a strict correlation between detergent resistance and apical targeting. Lubrol resistance is an intrinsic property of NPP3, which is acquired early during the biosynthetic process irrespective of its final destination, and depends on positively charged residues in its cytoplasmic tail.
Jennifer A Corcoran - One of the best experts on this subject based on the ideXlab platform.
-
the p14 fusion associated small transmembrane fast protein effects membrane fusion from a subset of membrane microdomains
Journal of Biological Chemistry, 2006Co-Authors: Jennifer A Corcoran, Jayme Salsman, Roberto De Antueno, Ahmed Touhami, M H Jericho, Eileen K Clancy, Roy DuncanAbstract:Abstract The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.
-
the p14 fusion associated small transmembrane fast protein effects membrane fusion from a subset of
2006Co-Authors: Jennifer A Corcoran, Jayme Salsman, Roberto De Antueno, Ahmed Touhami, M H Jericho, Eileen K Clancy, Roy Duncan, Fromthedepartmentsof MicrobiologyandimmunologyandAbstract:The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WXand Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.
Michèle Maurice - One of the best experts on this subject based on the ideXlab platform.
-
1 Differential solubilization of inner plasma membrane leaflet components by
2009Co-Authors: Triton X, Jean-louis Delaunay, Germain Trugnan, Michèle Maurice, Michelyne Breton, Inserm U, Faculté De Médecine Saint-antoineAbstract:A commonly-used method for analyzing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4°C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and inserm-00418165, version 1- 17 Sep 2009 Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.3 1
-
Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100.
Biochimica et Biophysica Acta:Biomembranes, 2008Co-Authors: Jean-louis Delaunay, Breton Michelyne, Germain Trugnan, Michèle MauriceAbstract:A commonly-used method for analyzing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4°C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.
-
Differential detergent resistance of the apical and basolateral NPPases: relationship with polarized targeting.
Journal of Cell Science, 2007Co-Authors: Jean-louis Delaunay, Germain Trugnan, Michelyne Breton, James Goding, Michèle MauriceAbstract:Targeting of glycosylphosphatidylinositol-anchored proteins to the apical surface of epithelial cells involves clustering in Triton X-100-resistant membrane microdomains or rafts. The role of these microdomains in sorting transmembrane proteins is more questionable because, unlike glycosylphosphatidylinositol-anchored proteins, apical transmembrane proteins are rather soluble in Triton X-100. They are, however, resistant to milder detergents such as Lubrol WX or Tween 20. It has been proposed that specific membrane microdomains, defined by resistance to these detergents, would carry transmembrane proteins to the apical surface. We have used MDCK cells stably transfected with the apical and basolateral pyrophosphatases/phosphodiesterases, NPP3 and NPP1, to examine the relationship between detergent resistance and apical targeting. The apically expressed wild-type NPP3 was insoluble in Lubrol WX whereas wild-type NPP1, which is expressed basolaterally, was essentially soluble. By using tail mutants and chimeric constructs that combine the cytoplasmic, transmembrane and extracellular domains of NPP1 and NPP3, we show that there is not a strict correlation between detergent resistance and apical targeting. Lubrol resistance is an intrinsic property of NPP3, which is acquired early during the biosynthetic process irrespective of its final destination, and depends on positively charged residues in its cytoplasmic tail.
Roberto De Antueno - One of the best experts on this subject based on the ideXlab platform.
-
the p14 fusion associated small transmembrane fast protein effects membrane fusion from a subset of membrane microdomains
Journal of Biological Chemistry, 2006Co-Authors: Jennifer A Corcoran, Jayme Salsman, Roberto De Antueno, Ahmed Touhami, M H Jericho, Eileen K Clancy, Roy DuncanAbstract:Abstract The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.
-
the p14 fusion associated small transmembrane fast protein effects membrane fusion from a subset of
2006Co-Authors: Jennifer A Corcoran, Jayme Salsman, Roberto De Antueno, Ahmed Touhami, M H Jericho, Eileen K Clancy, Roy Duncan, Fromthedepartmentsof MicrobiologyandimmunologyandAbstract:The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WXand Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.
-
The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains
ScholarWorks @ UTRGV, 2006Co-Authors: Corcoran, Jennifer A., Roberto De Antueno, Salsman Jayme, Touhami Ahmed, Jericho, Manfred H., Clancy, Eileen K., Duncan RoyAbstract:The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX-and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc
