Fig. 7.

Stalk-buttress interactions are essential for dynein motion. a Antibody-based dimerization of WT Dyn1_331kDa results in processive runs under non-reducing conditions in the single-molecule TIRF assay at 1 mM ATP (left). Diagonal lines in the kymograph represent dimerized dynein molecules that are moving over time. Fitting the histogram of measured velocities with a Gaussian (black line, top right), results in a mean velocity of 108 ± 2 nm/s (±SEM; N = 255). A histogram of measured run lengths fit with an exponential function (black line, bottom right) yields a characteristic run length of 1.1 ± 0.1 μm (±SEM; N = 255) (source data are provided as a Source Data file). b In contrast to the motors with the cross-linked β and γ stalk registrations (d), Dyn1_331 kDa-α CL shows highly processive motion (left) (the dashed white line serves as a visual guide to help recognize the slow moving Dyn1_331kDa-α CL mutant) under non-reducing conditions. Fitting the histogram of measured velocities with a Gaussian (black line, top right), yields a mean velocity of 10.6 ± 0.4 nm/s (±SEM; N = 336) (top right). Performing the experiments in the presence of 2 mM TCEP increases the velocity to 109 ± 3 nm/s (±SEM; N = 281) (bottom right), demonstrating the reversibility of the cross-linking of the stalk helices (source data are provided as a Source Data file). c Kymograph analysis of the antibody-dimerized motors with the buttress truncations reveals that neither ΔBUT1-Dyn1_331 kDa (left) nor ΔBUT-E3197K-Dyn1_331 kDa (right) move along MTs. Instead, both motors show non-motile MT interactions (increasing the ionic interaction strength between MTs and the MTBD via the E3197K mutation increases the lifetime of the non-motile binding events, see Supplementary Fig. 9b). d Kymograph analysis of the antibody-dimerized WT Dyn1_331 kDa-β CL and WT Dyn1_331kDa-γ CL motors reveals only non-motile MT interactions. e Preventing stalk-buttress interactions in the ΔBUT-Dyn1_331 kDa-α CL motor results in non-motile interactions, demonstrating the importance of stalk-buttress interactions. The depicted scale bars are the same for all kymographs shown in this figure