Fig. 3
Assembly of YFP-MreB at a planar membrane in vitro. a Preparation of membrane vesicles, stained with FM4–64 (red fluorescent dye), b Calcium-induced fusion of vesicles establishes a flat membrane on top of a glass slide. Membrane stain in red, green channel shows background fluorescence in the YFP channel. c Diffuse localization of monomeric YFP-MreB on the membrane after washing with buffer. Note the small hole in the membrane in the upper left corner. d Addition of magnesium (10 mM) induces the formation of YFP-MreB filaments (2 μM) at the membrane. e STED images of individual YFP-MreB filaments (2 μM, 10 mM MgCl2, no KCl), and examples of branching and fusion of filaments. f-i Different concentrations of YFP-MreB as indicated (10 mM MgCl2, no KCl). j In vitro fluorescently labeled MreB carrying an additional cysteine residue integrated into the N-terminus of the protein (Cys-MreB) (2 μM, 10 mM MgCl2), (k) low concentration of YFP-MreB mixed with Cys-MreB, (l) Mixture of a low concentration of YFP-MreB and a high concentration of non-fluorescence-tagged MreB. Note that all constructs carry a Strep-tag for purification. White bars 2 μm