Fig. 1.

Formation of invaginations and ILVs in GUVs by PFN. (A) Invaginations and secondary vesicle formation induced by 12 nM PFN in the presence of 0.1 mM Ca²⁺. The fluorescence derived from rDHPE is shown in red. (B) Time course of PFN-induced secondary vesicle formation. The formation of two invaginations on a single GUV may be seen, denoted by an arrow and an arrowhead. rDHPE fluorescence (red), D70 fluorescence (green), and a merged image is shown for the final image. The time (in seconds) after the mixing of all components is shown on each image. (C) Secondary vesicles are filled with external medium that contains fluorescent probes as indicated. For D4 also the primary GUV is full, because PFN pores formed on the membranes allow the passage of this small dextran across the membrane. (D) Three-dimensional reconstitution of GUVs with secondary vesicles. D70 was in the external medium. The indicated area is shown enlarged below. (E–F) Experiments were performed in the presence of 70 kDa FITC-labeled dextran, 12 nM PFN, and the amount of GUVs with secondary vesicles was quantified after 45 min, except as indicated. In total four to eight independent experiments were performed for each condition with 104–211 GUVs analyzed. Different conditions were compared with a nonparametric Mann–Whitney test (*, p < 0.05; **, p < 0.01; n.s., not significant). (E) Experiment was performed in the presence of 5 mM EDTA or 0.25 mM CaCl₂, as indicated. The buffer used for the purification of PFN was used as a control in the absence of PFN. Each data point represents an independent experiment, the median is shown by the black line. (F) PFN concentration dependence of ILV formation. Experiments that were performed in the presence of 6, 12, or 48 nM PFN are compared to the bovine serum albumin control (12 nM) and control where PFN (12 nM) was denatured with heat (dPFN). Quantification of ILVs was done for 12 nM PFN also after 10 min as indicated.