The most common method to modify cellular cholesterol content in vitro is to incubate cells with cyclodextrin (CD). However, it is not well understood how cholesterol is removed by CD. Recent development of protein probes that bind cholesterol allowed to visualize and to follow cellular cholesterol semi-quantitatively. In this image, non-toxic cholesterol-binding D4 fragment of bacterial toxin, perfringolysin O was conjugated with mCherry (mCherry-D4) and the protein was transiently expressed in HeLa cells for 48 h. Cells were synchronized to mitosis and incubated with and without 1 mM methyl-beta-CD (MbCD) in the presence of EGFP-D4 in the medium. Selected images are shown below.
The results suggest that the removal of outer leaflet cholesterol by MbCD is accompanied by the rapid flip of cholesterol from the inner leaflet to the outer leaflet. The authors, however, cannot exclude the possibility that MbCD preferentially removed cholesterol from the inner leaflet of HeLa cells.
Read more and see the supplemental videos:
Abe M, Kobayashi T (2021) Imaging cholesterol depletion at the plasma membrane by methyl-β-cyclodextrin. J. Lipid Res. 62 100077. https://doi.org/10.1016/j.jlr.2021.100077