Figure 1.

Calcium-dependent-permeabilization of dioleylphosphatidylcholine Giant Unilamellar Vesicles by ACT DOPC GUVs by ACT toxin. (A) Representative images of GUVs (black, if empty) in a solution of Alexa-Fluor488 (green) incubated in the absence or presence of 200 nM ACT and buffer without or with 10 mM CaCl₂ (left panel). Images were taken 30 min after mixing the components. The internalization of Alexa-Fluor-488 to the lumen of GUVs (green) corresponds to permeabilized vesicles. GUV composition was DOPC. In the right panel the total percentage of permeabilization after 30′ for each condition (± ACT ± CaCl₂) is depicted. In this case the threshold filling value to discriminate between permeabilized and non-permeabilized vesicles has been the 40%. (B) Distribution of the degree of filling of individual GUVs to Alexa-Fluor-488 (right panel), after treatment with 200 nM ACT for 30 min in buffer with 10 mM CaCl₂. The degree of filling was calculated for each individual vesicle, from confocal images as the one shown in the left pane. In each of three experiments, a minimum of 250 vesicles were analyzed per condition. Error bars represent S.D.