Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking

Area of Science:

• Molecular Motor Proteins
• Cellular Motility
• Biophysics

Background:

• Cytoplasmic dynein is a crucial dimeric motor protein responsible for processive movement along microtubules.
• The motor domain of dynein hydrolyzes ATP, inducing conformational changes that drive stepping motion.
• Understanding the precise stepping mechanism of dynein is essential for comprehending intracellular transport.

Purpose of the Study:

• To visualize the load-free stepping motion of processive dynein with high temporal and spatial resolution.
• To investigate the step sizes and probabilities associated with dynein's movement along microtubules.
• To elucidate the underlying mechanism of dynein's processive motility at the single-molecule level.

Main Methods:

• Utilized scattering imaging of gold nanoparticles (AuNPs) to track dynein stepping motion.
• Employed an artificially dimerized chimeric dynein construct labeled with a 30 nm AuNP on one head.
• Achieved 100 μs time resolution and sub-nanometer localization precision at 1 mM ATP concentration.

Main Results:

• Observed distinct 8 nm forward/backward steps and 5 nm side steps, correlating with microtubule binding cleft geometry.
• Found the probability of forward steps to be 1.8 times higher than backward steps, with similar probabilities for side steps.
• Limited evidence for one-head bound states suggested stepping is governed by a single rate constant.

Conclusions:

• Dynein primarily moves via small, biased stepping motions, with a slight suppression of backward steps.
• The observed stepping behavior is consistent with dynein's interaction with the αβ-tubulin dimer on microtubules.
• This study provides key insights into the fundamental mechanics of dynein-mediated processive transport.