Fluorescence recovery after photobleaching to study the dynamics of membrane-bound proteins in vivo using the drosophila embryo

Abstract

The epithelial-to-mesenchymal transition is a highly dynamic cell process and tools such as fluorescence recovery after photobleaching (FRAP), which allow the study of rapid protein dynamics, enable the following of this process in vivo. This technique uses a short intense pulse of photons to disrupt the fluorescence of a tagged protein in a region of a sample. The fluorescent signal intensity after this bleaching is then recorded and the signal recovery used to provide an indicator of the dynamics of the protein of interest. This technique can be applied to any fluorescently tagged protein, but membrane-bound proteins present an interesting challenge as they are spatially confined and subject to specialized cellular trafficking. Several methods of analysis can be applied which can disentangle these various processes and enable the extraction of information from the recovery curves. Here we describe this technique when applied to the quantification of the plasma membrane-bound E-cadherin protein in vivo using the epidermis of the late embryo of Drosophila melanogaster (Drosophila) as an example of this technique.