Fig. 2: A polyvalent virus-like-particle lipidic-receptor system for endocytosis.

A Schematic representation of the synthetic system. Shown is a genetically encoded nanoparticle (GEM) assembled from 180 copies of the encapsulin protein (dark green) coupled to GFP (light green) scaffold. A GPI-anchored anti-GFP nanobody (purple) inserted into the membrane (beige) serves as receptor. B Fluorescence micrograph of GEMs binding to the cell membrane of CV1 cells. Scale bar is 10 µm. Insets: (upper) magnified region of the GEM-GFP decorated membrane from the overview emphasizing monodisperse binding. A single particle is marked with a box. Scale bar is 2 µm. (lower) Transmission electron micrograph of purified GEM. Scale bar is 15 nm. Experiments have been repeated twice with similar results. C Fluorescence micrograph of GEMs bound to Giant Plasma Membrane Vesicles (GPMVs) of CV-1 cells expressing GPI-anchored nanobody. Cells were incubated with 0.45 nM GEMs 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 bar is 2 µm. D Fluorescence micrograph of GEMs bound to energy-depleted CV1 cells expressing GPI-anchored anti-GFP nanobody that were starved of cellular energy by 30 min incubation in starvation buffer (PBS+/+ supplemented with 10 mM 2-deoxy-D-glucose and 10 mM NaN3) followed by 1 h incubation with 2 µg of purified GEMs in starvation buffer 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 the inset. E Fluorescence micrograph of GEMs bound to CV1-cells expressing Clathrin-light-chain-dsRED incubated for 10 min with 2 µg of GEMs before live imaging on a TIRF microscope. Experiments have been repeated three times with similar results. Scale bars are 5 µm and 1 µm for inset. F Fluorescence micrograph of GEMs bound to CV1-cells expressing Caveolin-1-mRFP incubated for 10 min with 2 µg of GEMs before live imaging on a TIRF microscope. Experiments have been repeated three times with similar results. Scale bars are 5 µm and 1 µm for inset. G Correlative confocal fluorescence platinum-replica electron microscopy micrographs of plasma membrane sheets generated after unroofing cells incubated with GEMs. Shown are 4 representative intracellular plasma membrane structures colocalizing with GEMs bound to the outside of cells that are neither positive for clathrin (as shown by antibody-staining) nor caveolae (based on distinct caveolae protein coat). Scale bars are 50 nm. Electron microscopy micrographs are on top, same field of view with correlative GFP fluorescence of the GEMs at the bottom. H Top: Example platinum replica electron microscopy micrographs of a typical clathrin-coated pit, caveola and clathrin/caveolin double-negative invagination. Bottom: Quantification of colocalization of GEM fluorescence with endocytic structures. Means ± S.D. for 6 cells from n = 2 independent experiments. Scale bar is 50 nm. Overview images are provided in Supplementary Fig. S2 and Supplementary Movie 2. Source data are provided as a Source Data file.