Method for cellular imaging of palmitoylated proteins with clickable probes and proximity ligation applied to Hedgehog, tubulin, and Ras

J Am Chem Soc. 2014 Mar 26;136(12):4544-50. doi: 10.1021/ja410068g. Epub 2014 Mar 17.

Abstract

Hedgehog protein undergoes post-translational palmitoylation, which is critical for its signaling activity during embryonic development and in adult tissues. Due to a lack of suitable imaging methods, the trafficking route of palmitoylated Hedgehog has remained unclear in secretory cells. Here, we report a novel method for imaging the subcellular distribution of palmitoylated forms of cellular proteins with high resolution. The method utilizes clickable chemical reporters to label the entire palmitoylated proteome, followed by proximity ligation on antibodies to the click-conjugated dye and the protein of interest to reveal the spatial localization of specific palmitoylated proteins, as exemplified by sonic Hedgehog, tubulin, and Ras. Palmitoylated sonic Hedgehog is found in the endoplasmic reticulum, the Golgi apparatus, and at the plasma membrane but not the endosomal system in Hedgehog-secreting cells. Palmitoylated tubulin is found along microtubule tracks and also partially associated with the plasma membrane, while palmitoylated H-Ras is visualized at various cellular locations including the plasma membrane, Golgi apparatus and endoplasmic reticulum. Our method is broadly applicable to imaging the palmitoylation of cellular proteins as well as other protein post-translational modifications that are detectable by clickable chemical reporters.

MeSH terms

  • Cell Membrane / metabolism
  • Click Chemistry*
  • Endoplasmic Reticulum / metabolism
  • Golgi Apparatus / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Hedgehog Proteins / chemistry
  • Hedgehog Proteins / metabolism*
  • Humans
  • Lipoylation*
  • Models, Molecular
  • Molecular Imaging / methods*
  • Molecular Probes / chemistry*
  • Protein Conformation
  • Protein Transport
  • Tubulin / chemistry
  • Tubulin / metabolism*
  • ras Proteins / chemistry
  • ras Proteins / metabolism*

Substances

  • Hedgehog Proteins
  • Molecular Probes
  • Tubulin
  • ras Proteins