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Probing Variations of Reduction Activity at the Plasma Membrane Using a Targeted Ratiometric FRET Probe
Tarushyam Mukherjee1, Sriram Kanvah1, Andrey S Klymchenko2
1Discipline of Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar, Gujarat 382355, India.
Abstract:
Plasma membrane (PM) is the turntable of various reactions that regulate essential functionalities of cells. Among these reactions, the thiol disulfide exchange (TDE) reaction plays an important role in cellular processes. We herein designed a selective probe, called membrane reduction probe (MRP), that is able to report TDE activity at the PM. MRP is based on a green emitting BODIPY PM probe connected to rhodamine through a disulfide bond. MRP is fluorogenic as it is turned off in aqueous media due to aggregation-caused quenching, and once inserted in the PM, it displays a bright red signal due to an efficient fluorescence energy resonance transfer (FRET) between the BODIPY donor and the rhodamine acceptor. In the PM model, the MRP can undergo TDE reaction with external reductive agents as well as with thiolated lipids embedded in the bilayer. Upon TDE reaction, the FRET is turned off and a bright green signal appears allowing a ratiometric readout of this reaction. In cells, the MRP quickly labeled the PM and was able to probe variations of TDE activity using ratiometric imaging. With this tool in hand, we were able to monitor variations of TDE activity at the PM under stress conditions, and we showed that cancer cell lines presented a reduced TDE activity at the PM compared to noncancer cells.
Insights
Researchers developed a novel probe to measure thiol disulfide exchange (TDE) activity at the plasma membrane. This tool revealed reduced TDE activity in cancer cells compared to non-cancer cells.
Area of Science:
- Cell biology
- Biochemistry
- Chemical biology
Background:
- The plasma membrane (PM) is crucial for cellular functions, involving numerous reactions.
- Thiol disulfide exchange (TDE) reactions are vital cellular processes occurring at the PM.
Purpose of the Study:
- To design and validate a selective probe for reporting TDE activity specifically at the plasma membrane.
- To investigate TDE activity variations in different cell types and under stress conditions.
Main Methods:
- Development of a novel probe, Membrane Reduction Probe (MRP), utilizing BODIPY and rhodamine linked by a disulfide bond.
- MRP exhibits fluorescence changes (aggregation-caused quenching, FRET) enabling ratiometric readout of TDE reactions.
- Application of MRP in cell models and live cells for ratiometric imaging of PM TDE activity.
Main Results:
- MRP successfully localized to the plasma membrane and reported TDE activity in a PM model.
- Ratiometric imaging with MRP demonstrated dynamic monitoring of TDE activity variations in live cells.
- Cancer cell lines exhibited significantly lower TDE activity at the plasma membrane compared to non-cancerous cells.
Conclusions:
- MRP is an effective tool for real-time monitoring of plasma membrane TDE activity.
- Reduced PM TDE activity is a potential characteristic distinguishing cancer cells from non-cancer cells.
- This probe facilitates further investigation into the role of TDE in cellular processes and disease states.
