A membrane-permeable small molecule biosensor accesses intractable cells and animals without genetic manipulation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Genetically encoded fluorescent biosensors are crucial for studying protein conformation dynamics in live systems.
• Genetic encoding presents limitations in organisms with poorly understood genomes or those sensitive to manipulation.
• Non-genetic approaches are needed to broaden biosensor applications in diverse biological contexts.

Purpose of the Study:

• To develop a membrane-permeable small molecule fluorescent biosensor for calmodulin activation.
• To create a non-genetically encoded tool for real-time monitoring of calmodulin dynamics.
• To demonstrate the utility of this new biosensor in various biological models.

Main Methods:

• Covalent linkage of trifluoperazine (binds active calmodulin) to an environment-sensing merocyanine dye to create CaMero.
• Application of CaMero via extracellular incubation or intravenous injection in mice.
• Real-time fluorescence intensity measurements to detect calmodulin activation.

Main Results:

• CaMero exhibited a 12-fold change in fluorescence intensity upon binding to activated calmodulin.
• Sensitive real-time reporting of calmodulin activity was achieved in various models.
• Observed calmodulin activation waves in peristaltic intestine, serum-stimulated fibroblasts, and foraminifera.

Conclusions:

• CaMero serves as an effective, non-genetically encoded fluorescent biosensor for calmodulin activation.
• This approach simplifies studies in animals and expands accessible organisms for biosensor research.
• CaMero holds potential for future applications in human studies.