Analysis of Planar Cell Polarity Complexes by Proximity Biotinylation in Xenopus Embryos.

Abstract

Understanding signaling processes operating in cells during development and disease requires extensive knowledge of protein interactions. Proximity-dependent biotinylation mediated by a promiscuous bacterial biotin ligase is a sensitive approach for evaluating protein interactions under physiological conditions. This technique allows for assessing protein association when conventional pull-down assays are not applicable due to high background or transient nature of the interaction. In contrast to many studies of proximity biotinylation in cultured cells, this protocol has been adapted to detect protein interactions in Xenopus embryos. Here, we apply this technique to evaluate planar cell polarity (PCP) complexes formed by Prickle3 and Vangl2, and show that Prickle3 fused to the N-terminal fragment of the biotin ligase from Aquifex aeolicus efficiently biotinylates Vangl2 in vivo. We present our step-by-step proximity biotinylation protocol that provides a reliable semiquantitative assay for protein interactions and highlights the use of Xenopus embryos as a model for biochemical studies.