(F) Schematics showing the interaction of the GRAM1b with the plasma membrane (PM) before and after sphingomyelinase (SMase) treatment. Left: At rest, subthreshold levels of accessible PM cholesterol, or limited presence of the co-distribution of accessible cholesterol and anionic lipids in the inner leaflet of the PM, are not sufficient to induce interaction of the GRAM domain with the PM. Right: Liberation of the sphingomyelin-sequestered pool of cholesterol by SMase treatment leads to an increase in accessible PM cholesterol in both inner and outer leaflets (as unsequestered cholesterol can flip-flop between leaflets), resulting in concomitant increase in the co-distribution of accessible cholesterol and anionic lipids, including phosphatidylserine (PS), beyond the threshold in the inner leaflet of the PM, and inducing PM recruitment of the GRAM1b as it detects such co-distribution in the PM. 