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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.