Abstract
Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput. Here, we describe a semi-automated system for large-scale in situ proximity ligation assays (isPLA), combining isPLA in microtiter wells with automated microscopy and computer-based image analysis. Phosphorylations and interactions are digitally recorded along with subcellular morphological features. We investigated TGF-β-responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells, and we relate these events to cell cycle progression and local cell crowding via measurements of DNA content and nuclear size of individual cells, and of their relative positions. We illustrate the suitability of this protocol to screen for drug effects using phosphatase inhibitors. Our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.
Highlights
Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in highthroughput
We use in situ proximity ligation assays (isPLA), automated microscopy, and computerassisted image analysis to characterize and relate phosphocomplex signaling with morphological features in millions of individual cells
The isPLA analysis uses pairwise antibody binding to trigger rolling-circle amplification, serving to generate rolling-circle products (RCPs) that reflect sites of proximal binding of the pairs of antibodies
Summary
Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in highthroughput. 1234567890():,; Cellular responses to stimulation or inhibition are reflected in the dynamics of protein interactions and posttranslational modifications (PTMs)[1,2,3,4] These dynamic events control signaling cascades and gene expression patterns. Monitoring and detailing such actions can reveal pathway-specific effects of disease or responses to targeted therapies[1,5]. Smad linker phosphorylation patterns have been connected with TGFβ-mediated invasion and metastasis[14,17] It remains unclear exactly how linker phosphorylations tune signaling by TGF-β. All these characteristics make linker-phosphorylated Smads important targets for single-cell analysis
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