Fig. 1. Measurement of clustering of fluorescent GM1 analogues in live cells.

a Chemical structures of the GM1 analogues used in this study. b Schematic depicting loss of anisotropy upon clustering. The circle represents the radius within which homo-FRET can occur if two or more dyes are present within this circle. In the absence of clustering, the dyes are far apart, and emission is polarized. When clustered, multiple dye molecules are within the FRET distance, resulting in homo-FRET. Generally, this results in depolarization and therefore, lower anisotropy, unless clustering occurs in a manner that the orientations of the dye dipole are more rigidly locked, resulting in increased anisotropy. c A total emission intensity image of a cell incorporated with fluorescent C16: 0 GM1 with specific regions of interest (ROIs) for anisotropy mapping. d Scatter plot of anisotropy vs total intensity for 100% fluorescently labeled GM1 C16:0 (solid red circles), 80% unlabeled GM1 C16:0 with 20% fluorescently labeled Alexa488-GM1 C16:0 (open red circles). e Scatter plot of anisotropy vs total intensity for 100% fluorescently labeled GM1 C16:1 (solid black squares), 80% unlabeled GM1 C16:1 with 20% fluorescently labeled Alexa488-GM1 C16:1 (open black squares). f Scatter plots of anisotropy vs total intensity for Alexa488-GM1 C16:1 (black squares) and Alexa488-GM1 C16:0 (red circles) under no perturbation (solid symbols) or mβcd treatment conditions (open symbols). g Scatter plots of anisotropy vs total intensity for Alexa488-GM1 C16:1 (black squares) and Alexa488-GM1 C16:0 (red circles) of intact membranes (solid symbols) and blebs (open symbols). h Scatter plots of anisotropy vs total intensity for Alexa488-GM1 C16:0 in the presence (solid red circles) and absence of PS (open red circles). Absence of PS abrogates active Alexa488-GM1 C16:0 clustering. In d–h, means ± standard deviation is shown. n = 20 cells over 4 independent experiments for d and e, n = 15 cells over 6 independent experiments for f–h.