Figure 1.|. Site-specific labeling and tracking of the MTBD of dynein.

| a, The schematic of yeast dynein dimer bound to the MT. (Insert) The atomic model of the MTBD shows the positions of unique cysteines (yellow) introduced after removing surface-exposed cysteines in Dyn_CLM. b, The denaturing gel images show that Dyn_CLM-Q3231C is labeled more efficiently than Dyn_CLM with all surface-exposed cysteines removed (F: fluorescence, C: Coomassie-stained). c, Dyn_CLM-Q3231C monomers were labeled with different dyes at their MTBD and heterodimerized via SpyCatcher-SpyTag at the tail. d, Kymographs show robust processive motility of dual-labeled dynein heterodimer, similar to WT dynein. Scale bars x: 10 µm, y: 100 s. e, Trajectories show FIONA tracking of dynein heterodimers labeled with a single LD655 dye at 2 µM ATP with 100 ms temporal resolution. The red lines show a fit for the step-finding algorithm. f, The histogram of steps taken along the MT on- and off-axis. Dashed lines show increments of 8 nm steps.