Tracking the subcellular fate of 20(S)-hydroxycholesterol with click chemistry reveals a transport pathway to the golgi [Cell Biology]

March 4th, 2014 by Peyrot, S. M., Nachtergaele, S., Luchetti, G., Mydock-McGrane, L. K., Fujiwara, H., Scherrer, D. E., Jallouk, A., Schlesinger, P. H., Ory, D. S., Covey, D. F., Rohatgi, R.

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. While the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol (20(S)-OHC) with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl-derivative of 20(S)-OHC has allowed us to directly visualize this oxysterol by attaching an azide-fluorophore through cycloaddition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. While previous models have proposed non-vesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.