Measurements of the Force-Dependent Detachment Rates of Cytoplasmic Dynein from Microtubules

Abstract

Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein's molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein's multiple AAA+ ATPase domains regulate dynein's attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.

Keywords: Cytoplasmic dynein; Fluorescence labeling; Microtubule immobilization; Microtubule motor proteins; Microtubules; Optical trapping; Optical tweezers; Recombinant proteins; Single-molecule assays; Yeast gene manipulation.