Fig. 3: GEMs are endocytosed and are trafficked through the endolysosomal pathway.
From: Adhesion energy controls lipid binding-mediated endocytosis
A, B Correlative fluorescence light microscopy and transmission electron microscopy of GEMs internalized in CV-1 cells. A Timepoint 1 h after binding. B Timepoint 6 h after binding. Each panel top from left to right: Fluorescence micrograph of GEMs; transmission electron micrograph of same region; correlative images. Each panel bottom left: Transmission electron micrograph of inset above. Each panel bottom right: Volumetric 3D-reconstruction of electron micrographs. GEMs emphasized in green, membrane emphasized in purple. Experiments have been repeated twice with similar results. Scale bars are 500 nm for overview and 100 nm for insets. C Fluorescence micrographs from a time-course experiments of endocytosis showing the distribution of GEMs in CV1 cells expressing anti-GFP nanobody and Lamp1-mRFP. Cells were incubated with 2 µg of GEMs for the indicated time points at 37 °C before live imaging on a spinning disk confocal microscope. Scale bars are 10 µm. D Quantification of colocalization between GEMs and Lamp1-mRFP and between GEMs and Rab7-mRFP from timepoints indicated in C Means ± s.e.m., n = 3 independent experiments. E Quantification of GEM endocytosis upon treatment with genetic (siRNA against clathrin-heavy-chain and expression of dominant negative Dyn2-K44A) or chemical inhibitors (Nystatin/Progesterone, BafilomycinA and Cytochalasin D) as compared to mock treatment of controls (Transferrin endocytosis for siRNA against CHC and overexpression of DynK44A; SV40 endocytosis for Nystatin/Progesterone, BafilomycinA and CytochalasinD). Mean fluorescence intensity ± S.D. was determined from flow cytometry measurements of 6811–27,733 cells from n = 2 independent experiments. Source data are provided as a Source Data file.
