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Single Particle Fluorescence Burst Analysis of Epsin Induced Membrane Fission

Fig 3

Kinetics of liposome fission are temperature dependent.

(a) Histograms of BAS analyzed 200 nm-diameter, TopFluor-labeled, (5%) PtdInsP(4,5)P2 Folch liposomes (red) and products of ENTH incubation at 23°C for 20 (blue), 60 (green), and 100 min (purple) after addition of 2 μM ENTH. At each time point, an aliquot was removed and placed on ice, and measurements were started within 1–2 min. Inset indicates resolution of small particles in a 10-fold dilution of each reaction. (b) Heat map representation of the fractional intensity for each reaction shown in (a). (c) BAS histograms generated from starting liposomes (red) and products of ENTH incubation at 37°C for 20 (blue), 60 (green), and 80 min (purple) after addition of 2 μM ENTH. (d) Heat-map representation of the fractional intensity for each reaction shown in (c). Additional time points are shown, for increased resolution. The effect of incubating liposomes in the absence of the ENTH domain for 60 min at 37°C or 100 min at 23°C is shown as an additional row, above the respective heat maps. The data shown for the experiments conducted at 23°C is representative of four experimental replicates. The data shown for experiments conducted at 37°C is representative of three experimental replicates.

Fig 3

doi: https://doi.org/10.1371/journal.pone.0119563.g003