Detailed Analysis of the Dynein Stepping Mechanism using Multicolor Tracking

Abstract

Cytoplasmic dynein is a motor protein that moves processively towards the microtubule minus end. To characterize dynein's step size at both limiting (5 µM) and saturating (1 mM) ATP concentrations, we have improved the temporal resolution of FIONA to 2 milliseconds and tracked the movement of single yeast cytoplasmic dynein motors, each labeled with one quantum dot. In contrast to kinesin, dynein's step size is highly variable, and backwards and sideways steps are frequently observed. As the tail domain takes ∼8 nm steps, the heads advance by taking both short (∼8 nm) and long (∼16 nm) steps. These data indicate that the heads do not move in a strictly alternating manner, as is the case with kinesin and myosin. To further characterize how the dynein heads move relative to each other, we have tracked the movement of both heads simultaneously. To precisely measure the head-head separation vector, we have developed novel small quantum dots (10 nm diameter) that specifically attach to each dynein head. The fluorescent signals of the differently-colored quantum dots attached to dynein are then registered with 2 nm precision. Using this technique, we find the heads to be widely separated, mostly in the off-axis, indicating that the two heads walk along separate filaments. Our two-color stepping data also show that the dynein heads advance mostly by taking alternating steps, as is the case with kinesin. However, the heads may also move independently of each other, and one head may take multiple steps before its partner moves forward. Both the leading head and the trailing head may initiate a step. Taken together, these unprecedented behaviors indicate that cytoplasmic dynein achieves processive movement via a mechanism unique among the molecular motors.