Image-based High-throughput Mapping of TGF-β-induced Phosphocomplexes at a Single-cell Level

Overview

Journal Commun Biol

Specialty Biology

Date 2021 Nov 13

PMID 34773084

Citations 1

Authors

Peter Lonn

Rasel A Al-Amin

Ehsan Manouchehri Doulabi

Johan Heldin

Radiosa Gallini

Johan Bjorkesten

Johan Oelrich

Masood Kamali-Moghaddam

Ulf Landegren

Affiliations

Soon will be listed here.

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.

Citing Articles

Monitoring drug-target interactions through target engagement-mediated amplification on arrays and in situ.

Al-Amin R, Johansson L, Abdurakhmanov E, Landegren N, Lof L, Arngarden L Nucleic Acids Res. 2022; 50(22):e129.

PMID: 36189884 PMC: 9825164. DOI: 10.1093/nar/gkac842.

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