An optogenetic method for the controlled release of single molecules.

Area of Science:

• Cell biology
• Optogenetics
• Molecular imaging

Background:

• Studying protein function at the single-molecule level in live cells is challenging.
• Controlling protein localization and release is crucial for understanding cellular processes.

Purpose of the Study:

• To develop a novel optogenetic system for the controlled release of single molecules within cells.
• To enable light-inducible delivery of functional proteins for single-molecule imaging and cellular function reconstitution.

Main Methods:

• Utilized a photocleavable protein to confine soluble and transmembrane proteins to the Golgi apparatus.
• Employed short light pulses to release proteins into the cytosol and plasma membrane in a dose-dependent manner.
• Applied the system to reconstitute ion channel function and induce signaling pathways.

Main Results:

• Achieved light-dependent delivery of functional proteins at levels suitable for single-molecule imaging.
• Successfully reconstituted ion conductance by delivering BK and LRRC8 channels.
• Induced NF-kB signaling in T lymphoblasts via controlled release of a signaling protein, demonstrating functional complex formation.

Conclusions:

• Developed a versatile optogenetic tool for precise, single-molecule protein manipulation in live cells.
• The system simplifies access to single-molecule microscopy and functional reconstitution of cellular pathways.
• This method opens new avenues for investigating cellular function at the molecular level.