Analyzing the Integrin Adhesome by In Situ Proximity Ligation Assay

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2217; p. 71
• Main Authors: Perrino, Brian A, Xie, Yeming, Alexandru, Cristina
• Format: Journal Article
• Language: English
• Published: United States 2021
• Subjects: Actin Cytoskeleton - metabolism; Actin Cytoskeleton - ultrastructure; Antibodies - chemistry; Extracellular Matrix - metabolism; Extracellular Matrix - ultrastructure; Focal Adhesions - metabolism; Focal Adhesions - ultrastructure; Gastric Mucosa - metabolism; Gastric Mucosa - ultrastructure; Humans; Image Processing, Computer-Assisted; Immunohistochemistry - methods; Integrin alpha Chains - chemistry; Integrin alpha Chains - metabolism; Integrin beta1 - chemistry; Integrin beta1 - metabolism; Microscopy, Fluorescence; Molecular Probes - chemistry; Molecular Probes - metabolism; Multiprotein Complexes - chemistry; Multiprotein Complexes - metabolism; Muscle, Smooth - metabolism; Muscle, Smooth - ultrastructure; Oligonucleotides - chemical synthesis; Oligonucleotides - metabolism; Protein Binding; Immunohistochemistry; Proximity ligation assay; Integrin; Immunofluorescent microscopy; Protein–protein interaction; Cancer
• DOI: 10.1007/978-1-0716-0962-0_7

The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.