Fig. 7

Cysteine-rich domain mediates cholesterol-dependent spike clustering. a Schematic representation of spike^1268_10A, with ten cysteine-to-alanine substitutions (red). b Cholesterol binding assays: Representative absorbance spectra demonstrate direct interaction with spike¹²⁶⁸ and spike^1268_10A. c Quantification of cholesterol binding to spike¹²⁶⁸ and spike^1268_10A. d The representative fields of spatial co-localization of spike¹²⁶⁸/spike^1268_10A protein (red) and cholesterol (green) on the plasma membrane (control, +MβCD, +MβCD-CHO, +MβCD + MβCD-CHO). Scale bars, 5 μm. Scale bar applies to all images in this panel. Corresponding fluorescence intensity profiles of spike¹²⁶⁸/spike^1268_10A protein (red) and cholesterol (green) along linear membrane regions in treated cell membrane are attached to the right of the fluorescence images. e Box plots and data points show comparative analysis of PCC for colocalization between spike variants (spike¹²⁶⁸ and spike^1268_10A, red) and cholesterol (green), across four cell membrane treatment groups corresponding to panel (d) from N ≥ 4 independent replicates. ROI = 23 (control), 42 (+MβCD), 53 (+MβCD-CHO), 43 (+ MβCD + MβCD-CHO). f Reconstructed SIM images illustrating cholesterol-modulated spatial distribution of spike^1268_10A clusters. Scale bars, 5 μm. Scale bar applies to all images in this panel. g Box plots and data points show the counts of clusters per μm² for the spike^1268_10A protein corresponding to panel (f) from N = 4 independent replicates. h Box plots and data points show the size of clusters (Feret’s diameter) corresponding to panel (f) from N ≥ 4 independent replicates. Cluster quantification and diameter measurements were performed using NIS-Elements software, with ≥20 randomly selected cells in each group. i. Representative fields of view of the fluorescent spots of ACE2-vesicles docking to corresponding spike-vesicles. The representative fields of view are docking events of ACE2-vesicles with corresponding spike-vesicles (0 or 20 mol% cholesterol). Scale bars, 10 μm. Scale bar applies to all images in this panel. j Box plots and data points show the docking probability corresponding to panel (i) from N = 4 independent replicates. In panel (c), statistical analysis was performed using t-test. In panels (g) and (h), statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. In panels (e) and (j), Statistical analysis was performed using two-way ANOVA followed by Sidak’s multiple comparisons test