(A) Effects of mutation of G187 on the property of the GRAM domain of GRAMD1b (GRAM1b) to sense transient expansion of the accessible pool of PM cholesterol. Indicated EGFP-tagged mutant versions of GRAM1b were expressed in GRAMD1 TKO HeLa cells and imaged under confocal microscopy with or without SMase treatment (100 mU/ml for 1 hour at 37°C) or with SMase treatment followed by 5 min treatment with Methyl-β-cyclodextrin (MCD) (5 mM) to assess their PM recruitment. The mean values (n = 20 cells from two independent experiments for untreated and SMase-treated conditions; n = 10 cells from one experiment for SMase & MCD-treated conditions) of the ratio of PM signals to the cytosolic signals, as assessed by line scan analysis, are presented for each condition as a heatmap. Amino acids are ranked according to Goldman, Engelman and Steitz (GES) hydrophobicity scale with the most hydrophobic amino acid on the top. 