Single‐cell Imaging of Wnt Palmitoylation by Porcupine

Abstract

Visualization of the subcellular distribution of palmitoylated protein is critical to the understanding of the molecular mechanisms regulating protein movement and function in a cell. Due to a lack of suitable techniques, the trafficking route of palmitoylated forms of the protein of interest has remained unclear. To address this unmet need, we have developed a method to image the palmitoylated form of the protein of interest with subcellular resolution using clickable bioorthogonal fatty acids and in situ proximity ligation. Using this imaging method, we have shown that palmitoylated Wnt3a can be visualized throughout the secretory pathway and trafficks to multivesicular bodies that act as export sites in secretory cells. We demonstrate that Wnt3a is palmitoylated by fatty acids 13–16 carbons in length at Ser209 but not at Cys77. We find that porcupine (PORCN) itself is palmitoy‐lated, and this modification partially regulates Wnt palmitoylation and signaling. The data here reveal the role of O‐palmitoylation in Wnt signaling and suggest another layer of cellular control over PORCN function and Wnt secretion. Our method is broadly applicable to imaging the palmitoylation of cellular proteins, such as palmitoylated Shh, tubulin or Ras, as well as other protein post‐translational modifications that are detectable by clickable chemical reporters.