Fig. 5. Efficient retrograde transport of TeNT H_C depends on multiple MT-based motors. (A-C) Distribution of speed values observed between two consecutive frames for TeNT H_C-Alexa488 carriers (interval = 5 seconds). Retrograde movement is conventionally shown as positive. (A) Effect of dynein inhibition on TeNT H_C transport. MNs were incubated with control medium (718 carriers, n=7 independent experiments; black bars) or with 1 mM EHNA (142 carriers; n=4; white bars). Note the drastic decrease in the frequency of fast retrograde speed movements and the increase in pauses and slow anterograde movements. (B) Effect of kinesin inhibition on TeNT H_C transport. Incubation of MNs with 10 µM AA (308 carriers; n=3; white bars) increases the frequency of pauses and of slow anterograde movements, and induces a decrease of high speed values which is less marked than after EHNA treatment. (C) Simultaneous treatment of MNs with EHNA and AA (57 carriers; n=2; white bars) has similar effects on TeNT H_C transport to EHNA alone. (D) The speed distribution profile of TeNT H_C carriers in rat MNs is best described by the sum of three Gaussian components, centred at 0, 0.53 and 1.15 µm/second (718 carriers; n=7). Quantification of these speed components in the presence and absence of motor inhibitors allowed us to analyse the contribution of different molecular motors to retrograde transport (E). EHNA (white bars) abolished the fast component and caused a correspondent increase of pauses, whereas the intermediate component remained unaltered compared with control cells (black bars). AA alone (hatched bars) increased the frequency of stationary periods while reducing the contribution of the fast component. Simultaneous treatment of cells with EHNA and AA (grey bars) led to the same effects observed with EHNA alone. Error bars represent mean values ± s.e.m.

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