Fig. 2: Visualizing the two-pore channel activity of TOM-CC.
a Typical image (N > 5.3 × 105) of a non-modified agarose-supported DIB membrane with TOM-CC channels under 488 nm TIRF-illumination. The white squares mark spots of high (SH), intermediate (SI), and low (SL) intensity (Supplementary Movie S1). The TIRF image has not been corrected by fluorescence bleaching or by a filter algorithm. b Fitting the fluorescence intensity profile of the three spots marked in (a) to two-dimensional Gaussian functions (Supplementary Movie S2). Red, yellow and green intensity profiles represent TOM-CC in SL, SI, and SH demonstrating Tom40 channels, which are fully closed, one and two channels open, respectively. Pixel size, 0.16 μm. c Fluorescence amplitude trace and (d) amplitude histogram of the two-pore β-barrel protein channel TOM-CC. The TOM-CC channel switches between SH, SI, and SL permeability states over time. Inserts, schematic of N. crassa TOM core complex with two pores open in SH (top), one pore open in SI (middle), and two pores closed in SL (bottom) (EMDB, EMD-37616); e Representative single-channel optical recordings and (f) amplitude histograms of the TOM-CC subunit Tom40 and OmpF. Tom40 and OmpF are completely embedded in the lipid bilayer. In contrast to the two-pore β-barrel protein complex TOM-CC, Tom40, and OmpF exhibit one permeability state over time only. Insert, structural model of N. crassa Tom40 (EMDB, EMD-3761) and E. coli OmpF (PDB, 1OPF) with open β-barrel pores. Data were acquired as described in Fig. 1c at a frame rate of 47.5 s−1. a.u. arbitrary unit.
