Imaging Cellular Protein Fatty Acylation

Wnts are secreted palmitoylated glycoproteins that are important in embryonic development and human cancers. Due to a lack of tools, studying the behavior of Wnts and other post‐translationally palmitoylated proteins has proven challenging. Over the past few years, there have been considerable advances in developing biochemical techniques for detecting palmitoylated cellular proteins with good sensitivity. Yet, methods that enable tracking the palmitoylated form of an individual protein in single cells have been lacking. Such approaches will be invaluable as they provide an extra layer of spatial information about the palmitoylated state of a protein and its intracellular dynamics. Here we describe a strategy which integrates click chemistry, proximity ligation and fluorescence microscopy to visualize the palmitoylated form of proteins with subcellular resolution. We applied the system to image Wnt proteins, thereby revealing new biological insights about Wnt fatty acylation, trafficking and its biochemical regulation. The system is also adaptable to a high throughput format, making it of potential use in drug discovery to screen for or validate modulators of Wnt palmitoylation that may interfere with Wnt‐driven cancers. Because the method developed here is modular in nature, it can be applied to imaging the palmitoylation of cellular proteins as well as other protein post‐translational modifications that are detectable by clickable chemical reporters.