FRAP Analysis of LET-23::GFP in the Vulval Epithelial Cells of Living Caenorhabditis elegans Larvae

Abstract

[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). Materials and Reagents 1. C. elegans strain zhIs038 [let-23::gfp; unc-119(+)] (Haag et al., 2014) Note: For information on maintenance of C. elegans: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html. 2. 4% agarose pads (for an example see http://www.wormatlas.org/agarpad.html by Monica Driscoll) 3. M9 solution with 20 mM tetramisole hydrochloride (anesthetic) 4. M9 buffer for C. elegans (see Recipes) Equipment 1. Standard microscopy slides and 18 x 18 cover slips 2. Zeiss LSM710 confocal microscope equipped with 458/488/514 nm argon laser 3. Computer with FIJI (ImageJ. National Institutes of Health) and appropriate Plugins for FRAP analysis (see below)