MINFLUX dissects the unimpeded walking of kinesin-1

Abstract

We introduce an interferometric MINFLUX microscope that records protein movements with up to 1.7 nanometer per millisecond spatiotemporal precision. Such precision has previously required attaching disproportionately large beads to the protein, but MINFLUX requires the detection of only about 20 photons from an approximately 1-nanometer-sized fluorophore. Therefore, we were able to study the stepping of the motor protein kinesin-1 on microtubules at up to physiological adenosine-5′-triphosphate (ATP) concentrations. We uncovered rotations of the stalk and the heads of load-free kinesin during stepping and showed that ATP is taken up with a single head bound to the microtubule and that ATP hydrolysis occurs when both heads are bound. Our results show that MINFLUX quantifies (sub)millisecond conformational changes of proteins with minimal disturbance.

Zeroing in on motor proteins The super-resolution microscopy technique MINFLUX enables localization of fluorophores using a minimal number of photons. Two studies now expand on the development and implementation of MINFLUX to track motor protein dynamics in vitro and in cells (see the Perspective by Fei and Zhou). Wolff et al . refined the precision of MINFLUX such that single-fluorophore tracking with nanometer precision was possible with only tens of photons. They tracked the movement of kinesin-1 on microtubules and were able to see individual 4-nanometer substeps and rotation of the protein during stepping in their analysis. Deguchi et al . applied MINFLUX with a labeling and tracking approach called motor-PAINT to monitor stepping of motor proteins on microtubules in living and fixed cells in both two and three dimensions. —MAF

A high-resolution MINFLUX microscopy approach reveals kinesin steps, substeps, and rotations.