Measurement of Junctional Protein Dynamics Using Fluorescence Recovery After Photobleaching (FRAP)

Abstract

Fluorescence Recovery After Photobleaching (FRAP) (Lippincott-Schwartz et al., 2003; Reits and Neefjes, 2001) was employed to determine dynamic properties of proteins localized at the ephitelial zonula adherens (ZA) (Kovacs et al., 2011; Otani et al., 2006). The proteins of interest were expressed in cells using a knockdown and reconstitution approach in which endogenous proteins were depleted by RNA interference (RNAi) and replaced by expression of an RNAi-resistant gene fused to GFP (Priya et al., 2013; Smutny et al., 2010; Smutny et al., 2011; Vitriol et al., 2007). By choosing expression levels of GFP-tagged proteins that were comparable to endogenous levels, we minimized transient overexpression artifacts due to overcoming regulatory mechanisms that directly affect protein dynamics (Goodson et al., 2010). Using this approach, junctional E-cadherin-GFP or GFP-Ect2 were subjected to FRAP analysis in small areas corresponding to the ZA using confocal microscopy (Priya et al., 2013; Ratheesh et al., 2012; Gomez et al., 2005; Trenchi et al., 2009). Although in principle this approach is similar in every case, bleaching conditions, acquisition parameters and analysis details might differ depending on the time scale of the recovery process (Lippincott-Schwartz et al., 2003). In this protocol we will describe the experimental procedure to perform FRAP experiments and how to optimize bleaching and acquisition conditions for optimal measurements of protein dynamics at cell-cell junctions.