Fig. 5: Statistical correlation between channel activity and lateral mobility of TOM-CC.
a Trajectories for various modes of TOM-CC mobility. Class I, diffusion interrupted by periods of transient anchorage (sites of transient anchorage circled); Class II, free diffusion; Class III, permanent anchorage. Moving particles in SH are shown in the trajectories in green; transiently or permanently tethered molecules in SI are shown in yellow. Data were acquired for t = 60 s at a frame rate of 47.5 s−1. b D(SI) as a function of D(SH) individually plotted for all TOM-CC molecules in DIB membranes supported by non-modified (dark blue and light blue, n = 64) and Ni-NTA modified agarose (dark red and light red, n = 123). Frequency histograms of D(SI), \(D\left({S}_{H}\right)\) and \(D\left({S}_{I}\right)\) are shown on top and right side, respectively. Three classes can be defined: a main class (I) of moving particles in SH while being transiently tethered at SI \(\left\{I\left.\left|D\left({S}_{H}\right) \, > \, {D}_{{\min }}\right.\,\cap\, D\left({S}_{I}\right)\,\le\, {D}_{{\min }}\right\}\right.\), a second class (II) of freely moving particles in SH and SI \(\left\{{II}\left.\left|D\left({S}_{H}\right) \, > \, {D}_{{\min }}\right.\,\cap\, D\left({S}_{I}\right) \, > \, {D}_{{\min }}\right\}\right.\) and a third class (III) of permanently tethered molecules in SI and SL \(\left\{{III}\left.\left|D\left({S}_{H}\right)\,\le\, {D}_{{\min }}\right.\,\cap\, D\left({S}_{I}\right)\,\le\, {D}_{{\min }}\right\}\right.\). c Example trajectories (top and Supplementary Movie S7) and state probabilities (bottom) of non-permanently and permanently tethered TOM-CC in DIBs supported by non-modified and Ni-NTA-modified agarose. The probability of being in state SH is higher for non-permanently (classes I and II, dark blue [n = 46] and dark red [n = 40]) than for permanently tethered molecules (class III, light blue [n = 18] and light red [n = 83]). The probability of being in state SI is significantly higher for permanently (class III) than for non-permanently tethered particles (classes I and II). This suggests that binding of Tom22 to Ni-NTA agarose below the membrane triggers closure of the TOM-CC channels. The data are represented as median; the confidence intervals are given between 15 to 85% and 30 to 70%. d Absolute state transition probabilities classified by bidirectional state transitions as SH ⇆ SI, SI ⇆ SL, and SH ⇆ SL. Diffusive TOM-CC molecules have a significantly higher transition probability for switching between SH and SI in DIBs supported by Ni-NTA-modified agarose (~5.3%) than in non-modified agarose membranes (~2.3%). This is consistent with the higher efficacy of TOM-CC-trapping by Ni-NTA-modified agarose compared to non-modified agarose. Classification of non-permanently and permanently tethered TOM-CC is shown at the left bottom.
