Combined Poltirf and Sub-Pixel Particle Tracking of Cytoplasmic Dynein Supports a Winch-Like Stepping Mechanism

Abstract

Cytoplasmic dynein is a molecular motor that carries cargos toward the minus end of microtubules. Dynein walks processively, but less regularly along microtubules than kinesin and is capable of taking both backwards and sideways steps. Structural dynamics of dynein's AAA+ ring domain during processive stepping are not well understood. We use a combination of polarized TIRF and sub-pixel particle tracking to measure the position and orientation of fluorescent nanorods rigidly attached to AAA5 and AAA6 of the individual dynein ring domains via biotin-NeutrAvidin linkage. We observe rotational changes of the ring and how they correlate with translocation steps. The dynein ring undergoes frequent, small rotations, typically less than 20 degrees, about twice as often as steps. Stepping and tilting both depend on ATP, although some ATP-independent rotations are observed. Rotations which accompany translocation steps have larger magnitudes than ones that are not correlated with steps. Not all steps are correlated with angle changes, either. A heterodimeric mutant construct, with one biotinylated ring carrying a quantum rod and one non-biotinylated dead-head ring walks slowly, indicating that the rod does not destroy motility. Our results are inconsistent with a purely powerstroke stepping mechanism, analogous to that of myosin which would predict larger angle changes tightly coupled to stepping, but instead they support a winch-like mechanism that involves bending of the coiled-coil stalk under intermolecular torque between the heads in the double-headed bound state. Supported by NIH Grant P015GM087253.