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Published on: October 27, 2020
[Study of TGF-β/Smad3 signal pathway using the technology of flurorescence resonance energy transfer]
Abstract:
The transforming growth factor-β1 (TGF-β1)/Smad3 signal pathway is related to mutiple physiological and pathological generation mechanism of human being. Up to date, however, the spacial and time information on the phosphorylated Smad3 is still unclear. In this study, the process of Smad3 phosphorylation was observed under the physiological state in the living cells. Firstly, the ECFP-Smad3-Citrine (Smad3 biosensor) fusion protein expression vector was constructed and identified. Then the Smad3 biosensor was transfected into 293T cells. The transfection efficiency and the expressions of fusion proteins were observed in 24 hours. Thirdly, Smad3 biosensor flurorescence resonance energy transfer (FRET) was observed with the inversion fluorescence microscope and measured by the MetaFlour FRET 4. 6 software. Smad3 biosensor transfection efficiency was nearly 40% and the fusion protein was seen under the fluorescence microscope. The FRET ratio of Smad3 biosensor in living 293T cells was decreased after 10 minutes incubation with the ligand of TGF-β1. The period of decreasing CFP and enhancing Citrine signals was about 300 seconds. With the technology of FRET, the TGF-β1/Smad3 signal pathway could be real time monitored dynamically under the physiological condition in living cells.
Insights
This study visualizes Smad3 phosphorylation in living cells, revealing dynamic changes in the transforming growth factor-β1 (TGF-β1)/Smad3 pathway. This provides real-time insights into cellular signaling mechanisms.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway is crucial for various physiological and pathological processes in humans.
- Current understanding of the spatiotemporal dynamics of Smad3 phosphorylation remains limited.
Purpose of the Study:
- To develop and utilize a biosensor for real-time monitoring of Smad3 phosphorylation in living cells.
- To investigate the dynamic changes in the TGF-β1/Smad3 signaling pathway under physiological conditions.
Main Methods:
- Construction and identification of a Smad3 biosensor (ECFP-Smad3-Citrine fusion protein).
- Transfection of the biosensor into 293T cells and observation of expression.
- Measurement of Förster Resonance Energy Transfer (FRET) using fluorescence microscopy to detect Smad3 phosphorylation.
Main Results:
- Successful construction and expression of the Smad3 biosensor in 293T cells with approximately 40% transfection efficiency.
- A decrease in the FRET ratio was observed within 10 minutes of TGF-β1 stimulation, indicating Smad3 phosphorylation.
- Dynamic changes in CFP and Citrine fluorescence signals occurred over approximately 300 seconds.
Conclusions:
- Fluorescence resonance energy transfer (FRET) technology enables dynamic, real-time monitoring of the TGF-β1/Smad3 signaling pathway in living cells.
- This approach provides valuable spatiotemporal information on Smad3 phosphorylation, advancing our understanding of cellular signaling.
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