Fig. 3: Lateral mobility correlates with the channel activity of TOM-CC. | Communications Biology

Fig. 3: Lateral mobility correlates with the channel activity of TOM-CC.

From: Spatiotemporal stop-and-go dynamics of the mitochondrial TOM core complex correlates with channel activity

Fig. 3

a Scheme for imaging both position and channel activity of single TOM-CCs. b Representative TIRF microscopy images of a non-modified agarose-supported DIB membrane with three TOM-CC molecules taken from a time series of 60 s. The square-marked spot displays lateral motion, interrupted by a transient arrest between t = 11.0 s and t = 17.0 s. The arrow-marked spot corresponds to a non-moving TOM-CC until t = 48.8 s. Afterwards, it starts moving. Both moving spots show high fluorescence intensity (SH); the non-moving spots display intermediate (SI) or dark (SL) fluorescence intensity (Supplementary Movie S3). c, d Fluorescent amplitude trace and corresponding trajectory of the square- and arrow-marked TOM-CC as shown in (b) highlighted for two different time windows. Plots on top shows the change of amplitude over time, and plots on the bottom show the respective spatiotemporal dynamics for the three states. Comparison of the trajectories of single TOM-CC molecules with their corresponding amplitude traces reveals a direct correlation between stop-and-go movement and open-closed channel activity. Lateral diffusion of TOM-CCs in the DIB membrane is interrupted by temporary arrest, presumably due to transient linkage to the underlying agarose hydrogel. Although weak intensity profiles in SI do not allow accurate position determination, the fluorescent spots disappear and reappear at the same spatial x, y coordinates (red stars). The higher amplitude (c top) between t = 17.1 s and t = 25.1 s is due to the overlap between two adjacent spots. Green, TOM-CC is freely diffusive in SH; yellow and red, immobile TOM-CC in SI and SL. e Fluorescent amplitude trace and corresponding trajectory of a single β-barrel subunit Tom40. The Tom40 channel exhibits only one permeability state and is subject to simple thermal movement in the membrane (Supplementary Movie S5). Tom40 does not show stop-and-go motion as with TOM-CC. All data were acquired as described in Fig. 1c at a frame rate of 47.5 s−1. A total of nTOM = 64 and nTom40 = 20 amplitude traces and trajectories were analyzed. a.u., arbitrary unit.

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