Visualization of single molecules of dynein in living cells. (a) Schematic of the protocol followed for the visualization of single molecules of dynein. (b) Montage of HILO images showing representative binding and unbinding events of a single fluorescent mDHC molecule (“[binding]” and “[unbinding]”). The single molecule is indicated with yellow arrowheads for the duration of the time it remains bound in the field of view. Time is indicated at the top right of each image in the montage. (c) Intensity histogram of single molecules of dynein with the Gaussian fits (gray line). The mean ± SD of the Gaussian distributions is indicated above the fits. (d) HILO image (left) and kymograph (right) of a cell expressing mDHC-GFP. Representative stationary, minus end–directed, and plus end–directed events are indicated with the white, teal, and magenta arrowheads, respectively, in the kymograph and in the insets below. (e) Plot of position versus time for the single-molecule events tracked, showing stationary events (gray), minus end–directed events (teal), and plus end–directed events (magenta). n = 329 tracks from ∼50 cells across three independent experiments. (f) Histogram with the residence time of dynein on the MT on the x axis and P (τ) = 1 − cumulative frequency on the y axis. The exponential fit (gray line) gave a mean residence time τres = ^1/$roffD$ ∼0.59 s. In b and d, “N” marks the location/direction of the nucleus.