Analyzing the Integrin Adhesome by In Situ Proximity Ligation Assay.
Authors Perrino BA, Xie Y, Alexandru C
Submitted By Submitted Externally on 1/10/2022
Status Published
Journal Methods in molecular biology (Clifton, N.J.)
Year 2021
Date Published
Volume : Pages 2217 : 71 - 81
PubMed Reference 33215378
Abstract 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.