Image-based high-throughput mapping of TGF-β-induced phosphocomplexes at a single-cell level

• Published in: Communications biology Vol. 4; no. 1; p. 1284
• Main Authors: Lönn, Peter, Al-Amin, Rasel A., Doulabi, Ehsan Manouchehri, Heldin, Johan, Gallini, Radiosa, Björkesten, Johan, Oelrich, Johan, Kamali-Moghaddam, Masood, Landegren, Ulf
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.11.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 13/21; 13/95; 14/1; 14/63; 631/1647/2163; 631/337/458/1733; 631/80/86/2368; 82/47; 96/34; 96/63; 96/95; Automation; Biology; Biomedical and Life Sciences; Cell cycle; Cell size; Cytokines; HaCaT Cells; Humans; Image processing; Image Processing, Computer-Assisted; Life Sciences; Morphology; Peptide mapping; Phosphorylation; Protein interaction; Protein Interaction Mapping; Proteins; Single-Cell Analysis; Smad2 protein; Smad4 Protein - genetics; Smad4 Protein - metabolism; Transforming Growth Factor beta1 - genetics; Transforming Growth Factor beta1 - metabolism
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-021-02798-4

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. To improve our ability to monitor cellular responses to e.g. cytokines or drugs, Lönn et al have developed a semi-automated system for large-scale in situ proximity ligation assays (isPLA) in HaCAT keratinocyte cells. Their 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.