Fig. 5: Organization of membrane microdomains by flotillin complexes.

a, b Tomographic slice (left) and corresponding 3D reconstruction (right) from representative tomograms of regions marked by flotillin complex fluorescence. Flotillin complexes localize to the cytosolic leaflet of the plasma membrane and endosomes, as well as the luminal leaflet of extracellular vesicles (a), and are also observed in close association with actin filaments (b). The boxed areas highlight clusters of flotillin complexes within extracellular vesicles (a) and endosomes (b). Animated views of a and b are provided in Supplementary Movies 2 and 3, respectively. Subcellular components are colored as indicated. c After NEM treatment, GPMVs were isolated from cells stably expressing F1-EGFP and F2-mCherry, and separated into coexisting Lo/Ld phases as the temperature was lowered from room temperature to 4 °C. DiD dye marks Ld phase. d Comparative lipidomics analysis of endogenous lipids in purified flotillin samples and the plasma membrane fraction. Data are presented as mean ± SD from two biological replicates with two technical replicates each (individual data points represented as dots). Two-sided unpaired t-tests with Welch’s correction were performed without multiple comparisons correction. **p < 0.01, ***p < 0.001, n.s.: not statistically significant. Complete statistical information for null hypothesis testing is provided in Supplementary Data 1. LPC lysophosphatidic acid, PS phosphatidylserine, PG phosphatidylglycerol, PC phosphatidylcholine, PE phosphatidylethanolamine, PI phosphatidylinositol, SPH sphingosine, Cer ceramide, SM sphingomyelin, CH cholesterol, DG diacylglycerol, TG triacylglycerol. e Proposed model of flotillin membrane microdomain. Each minimal microdomain unit consists of a flotillin complex together with recruited partner proteins and sphingosines. The unit can cluster with others, driving lateral segregation within the membrane. Through this progressive clustering, larger functional membrane microdomains are ultimately formed. Tomographic slices are 1.2 nm thick. Scale bars, 120 nm (a, b), 10 µm (c).