A Chemically Switchable Synthetic Condensate Platform for Reversible Protein Sequestration and Release.

Area of Science:

• Synthetic biology
• Cellular engineering
• Molecular biology

Background:

• Controlling protein function in mammalian cells is crucial for understanding cellular processes.
• Artificial organelles offer a promising strategy for precise protein regulation.
• Existing methods often lack rapid, reversible control.

Purpose of the Study:

• To develop a synthetic condensate system for chemically controlled protein sequestration and release.
• To enable temporal and reversible regulation of cellular functions.
• To demonstrate the platform's utility in modulating both exogenous and endogenous proteins.

Main Methods:

• Constructed a synthetic condensate using phase-separated oligomeric proteins.
• Integrated a trimethoprim (TMP)-responsive nanobody switch (^GFPLAMA) for GFP-tagged proteins.
• Utilized TMP to induce rapid protein release from condensates and washout for re-sequestration.
• Applied the system to control membrane ruffling (GFP-Vav2) and ERK2-GFP localization.

Main Results:

• Successfully developed a synthetic condensate system for reversible protein sequestration and release.
• Demonstrated rapid, TMP-induced release of GFP-tagged proteins.
• Achieved user-defined temporal control over cellular processes.
• Showcased modulation of exogenous GFP-Vav2 and endogenous ERK2-GFP localization.

Conclusions:

• The ^GFPLAMA-based synthetic condensate platform provides a novel, chemically switchable tool for protein regulation.
• This system enables precise, temporal, and reversible control of cellular functions in mammalian cells.
• The platform holds significant potential for diverse applications in cell biology and biotechnology.