Figure 1.
Characterization of the direct insertion of γ-secretase into phase-separated planar-supported membrane systems with atomic force microscopy (AFM). (a) Schematic representation of the experimental procedure: direct insertion followed by lipid domain reorganization and then annealing and phase coalescence. (b−e) Time course of the direct insertion of γ-secretase into preformed DOPC/sphingomyelin/cholesterol (2:2:1)-supported bilayers on a glass-supported mica sheet. (b) Image at 25 °C prior to protein addition, and the image line profile below was obtained from the indicated line segment bisecting the image. (c) Image at 37 °C immediately after the addition of solubilized γ-secretase and the image line profile. (d) Image at 37 °C at t = 18 min after the addition of solubilized γ-secretase, and the boxed regions indicate where initial direct insertion has occurred. The three-dimensional (3D) image inset is zoomed in to show the 3D topography of an insertion event (scale bar 100 nm). (e) Image at 31 °C t = 37 min after the addition of solubilized γ-secretase and the image line profile. (f) Image (zoomed out to 10 × 10 μm2) at 25 °C after the addition of γ-secretase after 120 min of incubation time and cooling and the image line profile.