Fig. 4: Visualization of the flotillin complex in situ.

a Jurkat cells stably expressing flotillin-1-EGFP and flotillin-2-mCherry exhibit colocalization at the caps or uropods, as visualized by fluorescence microcopy. b Correlative cryo-light and electron microscopy workflow. Regions of interest identified by fluorescence microscopy serve as guide for subsequent tomographic tilt series collection by cryo-EM. c A 1.2 nm thick tomographic slice acquired within the area containing the GFP signal (corresponding to flotillin complexes). d 3D rendering of the tomogram shown in c. Flotillin complexes, plasma membranes, vesicles, ER and microtubules are colored as indicated. Selected flotillin complexes magnified in e are indicated by numbers. See Supplementary Movie 1 for animated visualization. e Tomographic slices zoomed in on the representative flotillin complexes identified within the tomogram shown in c. Notably, the side views of these complexes exhibit variability in shape. Additionally, some complexes (3 and 4, for example) contain internal regions of increased electron density. f, g Subtomogram averaging result of flotillin complexes (f) and its vertical cross-section views (g) at different contour levels. The model of flotillin complexes built with SPA map was fitted in to the STA maps for comparation. Scale bars, 20 µm (a), 5 µm (left panel in b), 1 µm (right panel in b), 120 nm (c) and 30 nm (e).