title: Proximity Ligation Assay (PLA) to Evaluate DISC and MISC Composition.
abstract: Distribution of the death receptor CD95 into lipid rafts (aggregation) and/or its internalization may contribute to the implementation of the apoptotic signal at the detriment of the non-apoptotic signaling pathway [1-6]. Also CD95 can form different protein complexes via dynamic protein-protein interactions (PPIs) according to its interaction with soluble or transmembrane CD95L. Therefore, spatiotemporal identification of these PPIs is pivotal to anticipate the signaling pathway implemented in cells stimulated with different forms of CD95L. Also, many disorders result from dysfunctions in terms of PPI subcellular distribution and/or their intensity, rendering evaluation of these features crucial to better understand pathogenesis.In situ proximity ligation assay (PLA) is a methodology that offers the possibility to identify PPIs and to determine where these PPIs occur in subcellular location (Fig. 1). Moreover, based on imaging, this method allows a quantification of PPIs at the cellular level and with a higher specificity than classical immunofluorescence assays. We here describe PLA used to confirm CD95/FADD interaction, a protocol that may serve to highlight other CD95 partners.
authors.affiliation: Centre Eugène Marquis, rue Bataille Flandres Dunkerque, 35042, Rennes, France.|INSERM ERL440-OSS, Equipe Labellisée Ligue Contre Le Cancer, Rennes, France.|Université de Rennes-1, 2 av. du Prof. Léon Bernard, 35043, Rennes, France.
authors.forename: Melissa
authors.initials: M
authors.lastname: Thomas
authors.affiliation: Centre Eugène Marquis, rue Bataille Flandres Dunkerque, 35042, Rennes, France. patrick.legembre@inserm.fr.|INSERM ERL440-OSS, Equipe Labellisée Ligue Contre Le Cancer, Rennes, France. patrick.legembre@inserm.fr.|Université de Rennes-1, 2 av. du Prof. Léon Bernard, 35043, Rennes, France. patrick.legembre@inserm.fr.
authors.forename: Patrick
authors.initials: P
authors.lastname: Legembre
mesh_terms: D017209:Apoptosis; D001681:Biological Assay / Q000379:methods*; D053222:Fas Ligand Protein / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction; D019014:fas Receptor / Q000378:metabolism
chemical_list: D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D008024:Ligands; D046912:Multiprotein Complexes; D019014:fas Receptor
keywords: CD95; Death inducing signaling complex; FADD; Protein–protein interaction; Proximity ligation assay