A fluorescent cholesterol analog, R-Chol, mimics the dynamics of free cholesterol in live cells

Cholesterol is an essential component of the mammalian cell membrane. Elucidating the dynamics of intracellular cholesterol is extremely important for understanding the mechanisms underlying life phenomena and diseases. The fluorescent cholesterol analog, R-Chol, has a fluorescent group at the 3-OH position of free cholesterol, which is less toxic and helpful for studying free cholesterol trafficking in live cells. In the present study, we determined the details of R-Chol dynamics in live cells. R-Chol was taken up by the plasma membrane within 10 min and then incorporated into the cells by endocytosis. Free cholesterol in late endosomes/lysosomes is transported to the endoplasmic reticulum via NPC1 proteins. In Npc1-deficient CHO cells, R-Chol accumulates in late endosomes/lysosomes. R-Chol accumulation in Npc1-deficient cells was attenuated by the overexpression of Rab9, which regulates intracellular transport from late endosomes to the Golgi apparatus. These results indicate that R-Chol incorporated via endocytosis is transported to late endosomes/lysosomes and then to the Golgi apparatus and endoplasmic reticulum by NPC1-dependent and NPC1-independent vesicular trafficking.