Free-cholesterol loading does not trigger phase separation of the fluorescent sterol dehydroergosterol in the plasma membrane of macrophages

Accumulation of excess non-esterified free cholesterol (FC) in macrophages is a key factor in macrophage death during late stages of atheroslerosis. Raising FC content in macrophages has been shown to trigger Rac activation and actin polymerisation and to inhibit cell migration. Here, the plasma membrane distribution of the fluorescent cholesterol-mimicking sterol dehydroergosterol (DHE) was investigated in FC-loaded J774 macrophages. Wide field fluorescence and deconvolution microscopy were combined with quantitative assessment of sterol distribution in straightened plasma membrane image segments. DHE's surface distribution matched exactly large ruffles and membrane protrusions which were pronounced in FC-loaded cells. Plasma membrane blebs, however, formed in FC-loaded J774 cells had a homogenous staining along the membrane bilayer at 20 degrees C. The results show that even in FC-loaded cells with increased membrane cholesterol content, sterols do not form a separate phase in the plasma membrane.