Image-based high-throughput mapping of TGF-beta-induced phosphocomplexes at a single-cell level
| Autor: | Masood Kamali-Moghaddam, Rasel A. Al-Amin, Ehsan Manouchehri Doulabi, Johan Björkesten, Peter Lönn, Johan Heldin, Radiosa Gallini, Ulf Landegren, Johan Oelrich |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
In situ
QH301-705.5 Cell Phosphatase Medicine (miscellaneous) Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology) Molecular Biology Microbiology Biochemistry or Biopharmacy) Article General Biochemistry Genetics and Molecular Biology Protein–protein interaction Transforming Growth Factor beta1 chemistry.chemical_compound Protein Interaction Mapping medicine Image Processing Computer-Assisted HaCaT Cells Humans Biology (General) Phosphorylation Throughput (business) Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi) molekylärbiologi mikrobiologi biokemi eller biofarmaci) Smad4 Protein Chemistry High-throughput screening Growth factor signalling Cell biology medicine.anatomical_structure Single-Cell Analysis General Agricultural and Biological Sciences Linker DNA Transforming growth factor |
| Zdroj: | Communications Biology, Vol 4, Iss 1, Pp 1-9 (2021) Communications Biology |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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