Fig. 3.

Inhibition of Dyn-2 activity impairs membrane resealing after laser injury. A: EA.hy.926 infected with adenoviruses as described above were incubated with FM1-43 (25 μM) and locally irradiated with a laser to induce membrane damage. Local increase in fluorescence was determined by 30-s time-lapse imaging for 10 min with a confocal microscope and analysis software. All AdK44A Dynamin-2 values, with the exception of origin, were significantly different (*) when compared with Adβ-Gal- or AdWT Dyn-2-infected cells. Experiments were performed on 6 EA.hy.926 per group in triplicate. B: representative images of a control (top) and an Ad K44A Dyn-2-treated (bottom) EA.hy.926 before, during, and 10 min after injury. Arrows show site of damage. Control cells show efficient resealing and expulsion of torn membrane structures outside of the plasma membrane. Deficient Dyn-2 activity decreases resealing after injury and causes patch accumulation close to injury site without efficient resealing. C and D: endogenous myoferlin colocalizes with Dyn-2 and lipid structures in EC by deconvolution fluorescence microscopy. Cultured EA.hy.926 were treated with a fixable FM1-43FX, which fluoresces in lipid structures (green), or a control solution for 1 h, rinsed, fixed and standard immunofluorescence (IF) against myoferlin (red) and Dyn-2 (green) was performed. Nuclei are shown in blue. Insets: typical merged nonimmune/control staining.