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- Title
Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells.
- Authors
Marvin, Dieuwke L.; You, Li; Bornes, Laura; van Dinther, Maarten; Peters, Niek; Dang, Hao; Hakuno, Sarah K.; Hornsveld, Marten; Kranenburg, Onno; van Rheenen, Jacco; Rohling, Jos H. T.; Chien, Miao-Ping; ten Dijke, Peter; Ritsma, Laila
- Abstract
Simple Summary: How a single cytokine can induce a variety of cellular responses in the same cell or in different cells is a longstanding question. Transforming growth factor β (TGF-β) is a prototypical multifunctional cytokine of which biological responses are highly dependent on in a cellular context. TGF-β signals via intracellular SMAD transcription factors, and the duration and intensity of SMAD activation are key determinants for the responses that are elicited by TGF-β. To visualize the TGF-β signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional reporter using a quickly folded and highly unstable green florescent protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β-induced responses in cells cultured on plastic dishes, and in living animals. This tool allows for the analysis of TGF-β signaling at a single living cell level, and allows for the discovery of dynamic TGF-β SMAD- induced transcriptional responses in multi-step biological processes. Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.
- Subjects
CELLULAR signal transduction; GENE expression; TUMORS; TRANSCRIPTION factors
- Publication
Cancers, 2022, Vol 14, Issue 10, p2508
- ISSN
2072-6694
- Publication type
Article
- DOI
10.3390/cancers14102508