Reversible Chemogenetic Fluorescence Labeling with pFAST in C. elegans

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Chemogenetic tools allow precise control of biological processes using small molecules.
• Fluorescence-based labeling is crucial for visualizing cellular structures and dynamics.
• Developing reversible and multicolor labeling systems is essential for complex biological studies.

Purpose of the Study:

• To generate and validate a tandem pFAST (td-pFAST) transgenic Caenorhabditis elegans strain for reversible chemogenetic labeling.
• To assess the feasibility of multicolor labeling using different fluorogens with the td-pFAST system.
• To investigate the limitations of fluorogen delivery in intact C. elegans.

Main Methods:

• Generation of a single-copy tandem pFAST transgenic C. elegans strain.
• Expression of td-pFAST in the pharyngeal muscle.
• Application of lime, amber, and coral fluorogens for fluorescence imaging.
• Testing reversibility using competing ligands (darth).
• Evaluation of fluorogen delivery methods (dissection vs. soaking).

Main Results:

• The td-pFAST system demonstrated rapid and reversible fluorescence labeling with lime fluorogen in dissected worms.
• Distinct emission spectra allowed for multicolor labeling with amber and coral fluorogens.
• Reversibility was confirmed by efficient quenching with a competing ligand.
• Fluorogen delivery via soaking intact worms was unsuccessful due to cuticle permeability barriers.

Conclusions:

• The td-pFAST probe is a functional tool for reversible, multicolor chemogenetic labeling in dissected C. elegans.
• The study highlights the potential of pFAST technology for advanced imaging in specific tissues.
• Cuticle permeability remains a significant challenge for in vivo applications in C. elegans.