Step Sizes and Rate Constants of Single-headed Cytoplasmic Dynein Measured with Optical Tweezers

Abstract

A power stroke of dynein is thought to be responsible for the stepping of dimeric dynein. However, the actual size of the displacement driven by a power stroke has not been directly measured. Here, the displacements of single-headed cytoplasmic dynein were measured by optical tweezers. The mean displacement of dynein interacting with microtubule was ~8 nm at 100 µM ATP, and decreased sigmoidally with a decrease in the ATP concentration. The ATP dependence of the mean displacement was explained by a model that some dynein molecules bind to microtubule in pre-stroke conformation and generate 8-nm displacement, while others bind in the post-stroke one and detach without producing a power stroke. Biochemical assays showed that the binding affinity of the post-stroke dynein to a microtubule was ~5 times higher than that of pre-stroke dynein, and the dissociation rate was ~4 times lower. Taking account of these rates, we conclude that the displacement driven by a power stroke is 8.3 nm. A working model of dimeric dynein driven by the 8-nm power stroke was proposed.

CaVβ controls the endocytic turnover of CaV1.2 L‐type calcium channel

“…Still, the range of Ca V 1.2 retrograde speeds is compatible with values reported for vesicular transport mediated by actin‐based myosin VI motor rather than by microtubule‐dependent dynein. Average speed values for mammalian cytoplasmic dynein determined with several techniques and ATP concentrations are up to an order of magnitude larger than the ones for myosin VI ranges 55‐61 . An actin‐operating motor protein controlling the retrograde transport of Ca V 1.2 is also consistent with the role of myosin VI in endocytosis and with recent work showing that Ca V 1.2 endocytic recycling is mediated by actin filaments, whereas being independent of microtubules 15,56,62‐64 …”

Quantitative Detection of Cell Activity by Measuring the Fluctuation of Intracellular Motility

“…Dimer kinesin (mouse KIF5A with 490 amino acid) or dynein (human dynein-1, GST-D384) labeled with BDTC were flowed to bind with streptavidin, and tubulin flowed. Purification of kinesin, dynein, and microtubules and labeling of the microtubules by rhodamine were carried out according to previous reports (Kinoshita et al, 2018). By adding 1 mM of ATP, the gliding of microtubules labeled with rhodamine was observed by fluorescence microscopy…”

A generalised mechano-kinetic model for use in multiscale simulation protocols

“…Brockwell et al also discovered that forced protein unfolding with an atomic force microscope yields different unfolding forces depending on the directions in which you apply that force, indicating dynamic anisotropy, and Berkovich et al showed that the collapse dynamics of unfolded proteins indicates force-dependent free energies of unfolding [31]. For cytoplamic dynein itself, a stalling force has been experimentally measured [11]. Thus, when under significant load or when opposed by other molecular motors such as kinesin [32], it may be the case that the instantaneous mechanical energy, not simply the average or minimum, plays a role in the overall kinetic cycle either by gating or accelerating certain kinetic transitions.…”

“…Experiments show that it is over much longer time scales that dynein undergoes its so called ‘powerstroke’, generating the directed force required for the motor to do the useful physical work of transporting cargo throughout the cytoskeletal network[11, 12, 13]. In vitro this powerstroke cycle, whilst constituted by cascading atomistic processes[14], is so slow compared to the dynamic time scales associated with the molecule that it is often modelled purely kinetically[15, 16].…”

“…showed that the collapse dynamics of unfolded proteins indicates force-dependent free energies of unfolding[31]. For cytoplamic dynein itself, a stalling force has been experimentally measured[11]. Thus, when under significant load or when opposed by other molecular motors such as kinesin[32], it may be the case that the instantaneous mechanical energy, not simply the average or minimum, plays a role in the overall kinetic cycle either by gating or accelerating certain kinetic transitions.…”