title: Combining immunofluorescence with in situ proximity ligation assay: a novel imaging approach to monitor protein-protein interactions in relation to subcellular localization.
abstract: The in situ Proximity Ligation Assay (PLA) is suited for visualizing protein-protein interactions and post-translational protein modifications in both tissue sections and in vitro cell cultures. Accurate identification and quantification of protein-protein interactions are critical for in vitro cell analysis, especially when studying the dynamic involvement of proteins in various processes, including cell proliferation, differentiation, and apoptosis. Here, we monitored the interactions between protein kinase-Cζ (PKCζ) and Bcl10 protein in untreated and etoposide (VP-16)-treated C4-I cells by means of a new combined morphological approach and validated it by taking stock of our previous proteomic and biochemical work (Chiarini et al. in J Proteome Res 11:3996-4012, 2012). We first analyzed the colocalization of PKCζ and Bcl10 proteins through classical immunofluorescent colocalization analysis. On the basis of these results, we developed a novel imaging approach combining immunofluorescence (IF) techniques with in situ PLA to identify the PKCζ·Bcl10 complexes at the level of a specific subcellular compartment, i.e., the nuclear envelope (NE). By this means, we could show that the amount of PKCζ·Bcl10 complexes localized at the NE of C4-I cells during proliferation or after treatment with VP-16 closely corresponded to our previous purely biochemical results. Hence, the present findings demonstrate that the combination of in situ PLA with classical IF detection is a novel powerful analytical tool allowing to morphologically demonstrate new specific protein-protein interactions at level of subcellular organelles, the complexes functions of which can next be clarified through proteomic/biochemical approaches.
authors.affiliation: Histology and Embryology Section, Department of Life and Reproduction Sciences, University of Verona Medical School, Strada le Grazie 8, 37134, Verona, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Pacchiana
authors.forename: Maurizio
authors.initials: M
authors.lastname: Abbate
authors.forename: Ubaldo
authors.initials: U
authors.lastname: Armato
authors.forename: Ilaria
authors.initials: I
authors.lastname: Dal Prà
authors.forename: Anna
authors.initials: A
authors.lastname: Chiarini
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000074018:B-Cell CLL-Lymphoma 10 Protein; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006801:Humans; D007834:Lasers; D018613:Microscopy, Confocal; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011493:Protein Kinase C / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000074018:B-Cell CLL-Lymphoma 10 Protein; D011493:Protein Kinase C; D000099221:Protein Kinase C zeta; C118591:BCL10 protein, human