Fig. 4: Adhesion energy controls membrane deformation and endocytosis of GEMs.

A Schematic representation of GPI-anchored nanobody constructs with decreasing binding affinity expressed in the outer membrane of cells used in this study. B Fluorescence micrographs of GEMs bound to GPMVs harvested from CV1 cells expressing the panel of GPI-anchored anti-GFP nanobody constructs as indicated and subsequently incubated with 0.45 nM GEM-GFP particles for 1 h at RT before imaging at the equatorial plane on a spinning disk confocal microscope. Experiments have been repeated twice with similar results. Scale bars are 2 µm. C Fluorescence micrographs of GEMs bound to energy-depleted CV-1 cells expressing the panel of GPI-anchored anti-GFP nanobodies. CV1 cells were starved of cellular energy by 30 min incubation at 37 °C in starvation buffer (PBS+/+ supplemented with 10 2-deoxy-D-glucose and 10 mM NaN3) followed by 1 h incubation with 2 nM of GEM-GFP particles in starvation buffer at 37 °C and imaged live on a spinning disk confocal microscope. DiI membrane dye was added 10 min prior to imaging at 1 mg/ml final concentration. Experiments have been repeated twice with similar results. Scale bars are 5 µm and 1 µm for insets. Arrows mark VLP-filled membrane invaginations. D Quantification of GEM-GFP endocytosis as a function of receptor affinity as determined by flow cytometry measurements of the mean cell-associated fluorescence after acid wash. Mean fluorescence intensity ± s.e.m. was determined from flow cytometry measurements of 846–9538 cells/sample from n = 3 independent experiments. E Quantification of GEM-GFP endocytosis as a function of receptor affinity and upon treatment with genetic (siRNA against clathrin-heavy-chain and expression of dominant negative Dyn2-K44A) or chemical inhibitors (Nystatin/Progesterone and BafilomycinA) as compared to mock treatment of controls (Transferrin endocytosis for siRNA against CHC and overexpression of DynK44A; SV40 endocytosis for Nystatin/Progesterone and BafilomycinA). Endocytosis was determined by flow cytometry measurements of the mean cell-associated fluorescence after acid wash. Mean fluorescence intensity ± S.D. was determined from flow cytometry measurements of 1026–29,803 cells from n = 2 independent experiments. Source data are provided as a Source Data file.