Fig. 1: Scheme for tracking single TOM-CC molecules and imaging their ion channel activity.

a TOM-CC was isolated from mitochondria of a Neurospora strain carrying a Tom22 with a hexahistidinyl tag (6xHis). Analysis of purified protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Blue staining revealed all known subunits of the core complex, Tom40, Tom22, Tom7, Tom6 and Tom5. The small subunits Tom7, Tom6 and Tom5 are not separated by SDS-PAGE. b Atomic model based on the cryoEM map of N. crassa TOM core complex (EMDB, EMD-3761⁶). The ionic pathway through the two aqueous β-barrel Tom40 pores is used to optically study the open-closed channel activity of individual TOM-CCs. Left, cytosolic view; right, side view; cis, mitochondria intermembrane space; trans, cytosol. Tom7, Tom6 and Tom5 are not labeled for clarity. c Single-molecule tracking and channel activity sensing of TOM-CC in DIB membranes using electrode-free optical single-channel recording. Left: Membranes are created through contact of lipid monolayer-coated aqueous droplets in a lipid/oil phase and a lipid monolayer on top of an agarose hydrogel. The cis side of the membrane contained Ca²⁺-ions (0.66 M), while having at the trans side a Ca²⁺-sensitive fluorescent dye (Fluo-8) and KCl (1.32 M) to balance the membrane osmotic pressure. The high Ca²⁺ content is necessary to induce high calcium flux through the TOM-CC pores and to provide reliable optical signals. Right: Ca²⁺-ion flux through individual TOM-CCs from cis to trans is driven by a Ca²⁺ concentration gradient, established around the two Tom40 pores, and measured by monitoring Fluo-8 emission in close proximity to the membrane using TIRF microscopy. Fluorescence signals reveal the local position of individual TOM-CCs, which is used to determine their mode of lateral diffusion in the membrane. The level of the fluorescence (high, intermediate and low intensity) correlates with corresponding permeability states of a TOM-CC molecule. A 100× TIRF objective is used both for illumination and imaging. Green dots, fluorescent Fluo-8; red dots, Ca²⁺ ions.