Cytoplasmic dynein transports cargos via load-sharing between the heads

Abstract

Cytoplasmic dynein is a motor protein that walks along microtubules (MTs) and performs mechanical work to power a variety of cellular processes. It remains unclear how a dynein dimer is able to transport cargos against load without coordinating the stepping cycles of its two heads. Here, by using a DNA-tethered optical trapping geometry, we find that the force-generating step of a head occurs in the MT-bound state, while the ‘primed’ unbound state is highly diffusional and only weakly biased to step towards the MT minus end. The stall forces of the individual heads are additive, with both heads contributing equally to the maximal force production of the dimer. Based on these results, we propose that the heads of dynein utilize a ‘load-sharing’ mechanism, unlike kinesin and myosin. This mechanism may allow dynein to work against hindering forces larger than the maximal force produced by a single head.

Molecular origin of the weak susceptibility of kinesin velocity to loads and its relation to the collective behavior of kinesins

“…4A), such that the velocity changes slowly at small forces, as shown in experiments (25). In contrast, for other motor proteins with a strong χ vel (F), such as myosins and dyneins, the velocity sharply diminishes at low external loads (26,27), following the lower curve (red in Fig. 4A).…”

“…Previous single-molecule analyses have shown that the kinesin motor velocity changes sluggishly at low loads [i.e., χ vel (F) is low below the single-kinesin stalling force] (25). In contrast, the velocities of single myosin and dynein motors decrease much more rapidly at low loads, yielding a stronger χ vel (F) in the same loading regime (26,27). Herein, we pinpointed the molecular origin of the weak χ vel (F) for kinesins by analyzing the impact of loading forces on the transition rates in the chemomechanical cycle.…”

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

“…At 8.3 nm, 16.6 nm, and 24.9 nm of separation, d t , between the MT-binding sites, the deflection sizes of the rings, d r , along the MTs are calculated to be 0.18 nm, 0.37 nm, and 0.55 nm, respectively, d t = d c + 2·d r = F c ·(1/k c + 2·C s ), and corresponding step sizes measured at the ring, s r = d c , are 7.9 nm, 15.9 nm, and 23.8 nm, thereby broadening the distribution of measured step sizes. Intramolecular forces at the three values of d t are 0.2 pN, 0.4 pN, and 0.6 pN, which are well within the 3-to 5-pN forcegenerating capability of the motor (43,46,53,54).…”

“…Ring rotations were also measured in the context of a heterodimer with one inactive or "dead" head ( Fig. 4 A and B), because it has been shown that a dynein construct having only a single active head is still able to move processively as long as the second head retains the ability bind to the MT (46). We used a heterodimeric construct in which the active head was doubly biotinylated and labeled with a QR, whereas the other unlabeled head contained a P-loop (Walker A) K-to-A mutation in AAA1, which renders it unable to bind or hydrolyze ATP (47).…”

Emerging roles of NudC family: from molecular regulation to clinical implications

“…It has two ~550 kD heavy chains with multiple ATP binding sites, three to four intermediate chains of ~74 kD, four light intermediate chains of ~55 kD, and several light chains of 8-22 kD (Nishikawa et al, 2014;Belyy et al, 2014;Bhabha et al, 2016). Cytoplasmic dynein plays essential roles in many cellular processes, as it can hydrolysis ATP to generate force to move on and towards the minus end of the microtubule (Karki and Holzbaur, 1999;Roberts et al, 2013).…”