(A) Tube pulling setup. A membrane tube is pulled from a GUV aspirated in a micropipette. Protein is injected using a second pipette. (B) Endophilin constructs used for the experiments. (C) Confocal images of tube after dynamin injection, as in setup (A). GUV is on the right, bead is on the left. (D) Confocal images of a tube after co-injection of endophilin (2x) dynamin (1x) and GTP. (E) Cumulative fission probability of tubes for various molar ratios of endophilin/dynamin, using 5 µM dynamin, 150 µM GTP. Lines: exponential fits to a*(1-exp(-t/τ)), n(−Endo)=15, n(+Endo (0.5x))=19, n(+Endo (1x))=18, n(+Endo (2x))=16, n(+Endo (4x))=15. Matlab code available in Source code file 1. (F) Fission efficiencies extracted from fits to data shown in (E). (G) Average fission time τ from fits to data shown in (E). (H) Cumulative fission probability for different endophilin constructs shown in (B) at 4x molar endophilin/dynamin ratio. n(+Endo (5 µM))=17, n(BAR)=16, n(SH3)=15. Lines: exponential fits to a*(1-exp(-t/ τ), Matlab code available in Source code file 1. (I) Fission efficiency extracted from fits to data shown in (H). (J) Average fission times τ extracted from fits to data shown in (H). Error bars in (F, G, I, J) indicate 95% confidence intervals of fits. (K–O) Decreased dynamin fluorescence density in co-complex with endophilin correlates with reduced fission efficiency. (K) Averaged dynamin fluorescence intensities for three different endophilin/dynamin ratios. The indicated values are calculated from integrals of the fluorescence peaks obtained following the image analysis explained in M. Source data are available in the Figure 2—source data 1. (L) Cumulative fission probability for the same tubes whose fluorescence was measured in (K). Matlab code available in Source code file 2. For (K–O), 150 µM GTP and 5 µM Dyn were used. (M) Image analysis of tubes coated with either dynamin alone or in co-complex with endophilin. From the dynamin image, a mask is generated (see magenta box), and a projection along the edge perpendicular to the tube axis is calculated (see vertical profiles). The intensity values shown in (K) are integrals of these vertical profiles. (N) Representative confocal images of dynamin fluorescence intensity signal for dynamin alone (−Endo) or in co-complex with endophilin (+Endo (1x)). (O) Vertical profiles obtained by image analysis shown in (M) of tubes shown in (N). Error bars in (K) indicate standard deviation. Scale bars are 5 µm, except in magenta box for (M), 1 µm.