Figure 1(A) Schematic of the in vitro FRET assay to detect co-distribution of phosphatidylserine (PS) and accessible cholesterol in membranes. Liposomes containing PS and cholesterol were mixed with an accessible cholesterol biosensor (ECFP-D4H) and a PS biosensor (mVenus-Lact-C2). ECFP-D4H was excited at 430 nm and emission from mVenus-Lact-C2 due to FRET between ECFP and mVenus was recorded at 525 nm. (B) Representative emission spectra of mixtures containing ECFP-D4H, mVenus-Lact-C2, and liposomes containing indicated compositions of lipids. Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%) and that FRET signal was decreased when sphingomyelin (SM) (25%) was additionally incorporated into the liposomes. Asterisks indicate positions of emission maximum of ECFP (*477 nm) and FRET (**525 nm). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; SM, sphingomyelin (N-oleoyl-D-erythro-sphingosylphosphorylcholine). (C) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm (FRET/ECFP) (see Materials and Methods) from mixtures as shown in (D) (mean ± SEM, n = 4 independent experiments; Dunnett's multiple comparisons test, **P < 0.0001). (D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing mCherry-GRAM1b. Cells were pre-incubated with either control imaging buffer (for control) or imaging buffer containing purified ECFP-D4H proteins (3 µM) (for masking accessible PM cholesterol) for 30 min at 37°C and imaged with the same buffer conditions. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 32 cells (Control), n = 35 cells (Pre-incubation w/ ECFP-D4H); data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001]. (E) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing EGFP-GRAM1b together with either mCherry for control or mCherry-Lact-C2 for PS masking. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 53 cells (mCherry), n = 32 cells (mCherry-Lact-C2 OE); data are pooled from four independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001]. (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.