Figure 4

Atomic force microscopy characterization of the mixing of ANXA4 and the crosslinking annexins. Schematic representation of AFM imaging of proteins structures that form when 1:1 annexin mixtures (ANXA4 and ANXA1-GFP (A), ANXA4 and ANXA2-GFP (E) and ANXA4-GFP and ANXA6 (I)) are added to DOPC/DOPS (1:1 molar ratio) supported lipid bilayers where the presence of the GFP label on provides a height differential between the two proteins. 1.5 μm scan on a mixture of ANXA1-GFP and ANXA4 indicates that the proteins do not mix (B). 250 nm scan (white square, B) allows ANXA4 trimers to be resolved (C). Fourier transform of (C) indicates a crystalline structure (D). Lattice vectors in Fourier space are indicated by arrows with corresponding vector length and period lengths noted in the same color. 1.5 μm AFM image of the protein structure that forms when a 1:1 mixture of ANXA4 and ANXA2-GFP is added to a supported lipid bilayer (shown schematically, E) (F). 250 nm scan (white square, F) allows ANXA4 trimers to be resolved (G). Fourier transform of (G) indicates a crystalline structure (D). Lattice vectors in Fourier space are indicated by arrows with corresponding vector length and period lengths noted in the same color. 500 nm AFM image of the protein structure that forms when a 1:1 mixture of ANXA4-GFP and ANXA6 is added to a supported lipid bilayer (shown schematically, I) (J). 250 nm image (L). Quantification of area occupied by ANXA4-GFP (red circles) and ANXA6 (purple circles) in a 1:1 mixture in 500 nm scans (N = 20, from 3 independent experiments) indicates that ANXA4-GFP occupies 30% of scan area and ANXA6 occupies 70% of scan area (M). Images prepared using JPK Data Processing Software, version 7.01.145 (https://www.bruker.com/de/products-and-solutions/microscopes/bioafm/jpk-nanowizard-4-xp-bioscience.html).