FRAP analysis of LET-23::GFP in the vulval epithelial cells of living caenorhabditis elegans larvae

Abstract

The Caenorhabditis elegans (C. elegans) vulva is a well-established system to study organ development as the molecular mechanisms that govern its formation are conserved in animals. Of special interest is the EGFR/RAS/MAPK signaling pathway that is required for fate acquisition and morphogenesis of the vulva. let-23 encodes the sole homologue of the epidermal growth factor receptor (EGFR), is expressed at the plasma membrane of the vulval precursor cells (VPCs) and is activated by LIN-3 EGF at the end of the L3 larval stage to initiate vulva development. LET-23 activity can be modulated through altering its subcellular and plasma membrane localization. To study the trafficking of EGF receptor in a living organism, we created a functional LET-23::GFP translational reporter worm line (Haag et al., 2014) and quantified the mobile fraction of LET-23::GFP at the basolateral membrane of the VPCs by fluorescence recovery after photobleaching (FRAP). Here we describe the protocol for LET-23::GFP FRAP at the basolateral membrane of the VPCs and the data analysis using FIJI (ImageJ).