CeLINC, a fluorescence-based protein-protein interaction assay in Caenorhabditis elegans

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Protein-protein interactions are crucial for cellular functions.
• Accurate methods are needed to study these interactions in vivo.
• Understanding protein interactions aids in deciphering protein function.

Purpose of the Study:

• To develop and validate a novel optical assay, Caenorhabditis elegans light-induced coclustering (CeLINC), for detecting protein-protein interactions in vivo.
• To assess the reliability and robustness of the CeLINC assay using reference protein pairs.
• To investigate interactions among apical and basolateral polarity regulators in C. elegans.

Main Methods:

• The CeLINC assay utilizes light-dependent CRY2/CIB1 oligomerization to induce artificial protein clusters.
• Fluorescently tagged bait proteins are captured and clustered, and prey protein colocalization indicates interaction.
• The assay was validated with positive and negative reference protein pairs and applied to study polarity regulators.

Main Results:

• The CeLINC assay demonstrated high robustness with no false positives detected in negative controls.
• The assay confirmed known interactions between PAR-6, PKC-3, and PAR-3.
• No physical interactions were detected among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1.

Conclusions:

• CeLINC is a powerful, rapid, and reliable optical assay for studying protein-protein interactions in vivo.
• The assay provides a flexible toolkit for C. elegans research, compatible with existing strains.
• CeLINC offers new possibilities for investigating protein complex formation and cellular processes.