Fig. 7: DAG facilitates membrane binding and tubulation by EndoB1.

a, b Representative images of a planar bilayer (magenta) containing 5% PI (a) or 5% DAG (b) with 15% CL, before and after incubation with EndoB1-mEGFP (green). c Representative images of a planar bilayer (magenta) of the indicated lipid composition after incubation with EndoB1^N-BAR (Residues 1–270)-mEGFP (green). White arrows in magnified panels mark protein clusters that coincide with regions of high membrane fluorescence indicating tubulation. d Representative images of membrane tubes (magenta) of the indicated lipid composition incubated with EndoB1^N-BAR-mEGFP (green). e Membrane densities of EndoB1^N-BAR-mEGFP are reported as the ratio of EGFP and membrane fluorescence across a range of tube sizes as well as on the planar bilayer island of the indicated lipid composition. Data are normalized to the membrane density of EndoB1^N-BAR-mEGFP on tubes of < 15 nm radius containing 5% DAG and 15% CL and represents the mean ± SD of 63 tubes and 31 different regions on planar bilayers for DAG-containing and 106 tubes and 35 different regions on planar bilayers for PI-containing membranes. Statistical significance was estimated using unpaired Mann-Whitney’s test. *** denotes p < 0.0001, ** denotes p = 0.074, * denotes p = 0.0238. f Representative time-lapse images of a planar bilayer responding to flowing EndoB1^N-BAR-mEGFP, which are also presented as Supplementary Movie 9. The inset shows a magnification of a single representative tubulation event. Images are acquired in the membrane fluorescence channel and are inverted in contrast for clarity. White arrows mark a single tubule, yellow arrows mark events of coiling of the tubule, and green arrows mark the coalescence of other single tubules in the vicinity. g Representative time-lapse images showing fluorescence recovery after photobleaching the intrinsic fluorescent lipid probe in a large area of the planar bilayer displaying EndoB1^N-BAR-mEGFP-coated tubules, which are also presented as Supplementary Movie 10. The region above the dotted line is where the lipid probe was bleached.