A protocol for single molecule imaging and tracking of processive myosin motors
Authors: Gardini L., Arbore C., Capitanio M., Pavone FS.
Autors Affiliation: European Lab Nonlinear Spect, LENS, Via Nello Carrara 1, I-50019 Sesto Fiorentino, Italy; Natl Inst Opt, Natl Res Council, Largo Fermi 6, I-50125 Florence, Italy; Univ Florence, Dept Phys & Astron, Via Sansone 1, I-50019 Sesto Fiorentino, Italy
Abstract: Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell’s life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins. (C) 2019 Published by Elsevier B.V.
Volume: 6 Pages from: 1854 to: 1862