Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

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. ABSTRACT: 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.