Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells. Cholesterol is an important component of biological membranes, but probes recording its dynamic intracellular distribution are scarce. Rakers et al. developed cholesterol-derived imidazolium salts mimicking properties of natural cholesterol. Following specific labeling via click chemistry, one of the cholesterol analogs was shown to incorporate into cellular membranes equivalent to endogenous cholesterol.