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

Abstract

The force-generating mechanism of dynein differs from the forcegenerating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.ynein is a molecular motor that walks processively toward the minus end of microtubules (MTs) in an ATP-dependent manner (1-3). Axonemal dyneins drive the motility of eukaryotic cilia and flagella, whereas cytoplasmic dynein is responsible for a wide range of functions within eukaryotic cells, including the retrograde transport of cargo such as autophagosomes in neurons (4), alignment of the mitotic spindle (5, 6), and chromosome segregation during mitosis (7). Disruption of dynein-mediated neuronal transport has been implicated in neurodegeneration (8), and mutations in dynein and dynein-associated proteins can cause a range of diseases, including spinal muscular atrophy (9, 10), lissencephaly (11) and Charcot-Marie-Tooth disease type 2 (reviewed in ref. 12). Despite the importance of dynein function, the mechanism by which dynein walks along the MT is not yet well understood.The motor domains of dynein are formed from six concatenated AAA domains, interrupted by a long, antiparallel, coiled-coil stalk that emerges from AAA4 and terminates in the microtubulebinding domain (MTBD) and the buttress that extends from AAA5 and is thought to stabilize the stalk (13,14). Two motor domains form a dimer via their N-terminal tails (2, 3). ATP binding and hydrolysis drive dynein mechanochemistry; the binding of ATP to AAA1 induces a conformational change leading to dissociation of the MTBD from the MT. Hydrolysis on the dissociated head induces a primed conformation that then rebinds to the MT. The forward step, or power stroke,...

Self-organized networks: Darwinian evolution of dynein rings, stalks, and stalk heads

“…The CC in AAA5 may act primarily as a buttress supporting the shaft, as CC2 has already hydrophobically reflected the ATP AA1 water wave signal back to the shaft head. A model showing small tilts of flexible stalks observed by polarized total internal reflection fluorescence microscopy is also consistent with this rocking model [30].…”

Diverse stepping motions of cytoplasmic dynein revealed by kinetic modeling

“…which is actually identical to the hypothetical model suggested previously [11,53]. In our kinetic model, however, this mode is sub-dominant.…”

“…In our model, this pathway is minor since the transition from 51DMDM-to 52TDM-occurs with a relatively low probability. A recent single-molecule assay measured the AAA+ ring angles relative to MT, finding that about 50% of motor domain steps are coupled with small angle changes in the AAA+ ring [52]. The Path 1 is perfectly in harmony with this result because only the leading motor domain takes the ATP hydrolysis cycle which exerts the force and leads to changes in the AAA+ ring angle.…”

“…However, the lifetimes of the pre-power-stroke states are rather short: From the populations in every states (S2 Table), we estimated the probability to have at least one pre-power-stroke state is only 15%, as a whole. Thus, due to short lifetimes of the pre-power-stroke states, some of these angle changes in the stalk might not been observed by the single-molecule assay [53].…”

Fundamental limits of measuring single-molecule rotational mobility

“…Since single molecules were first observed almost 30 years ago, 1 their rotational dynamics have been used to reveal the nanoscale organization of DNA [2][3][4] and the movement of molecular motors. [5][6][7][8][9] To discern molecular rotation, the fluorescence emitted by single molecules can be phase-modulated, detected after polarization filtering, and/or measured in response to the polarization of the excitation light. 10 Since fluorescence intensity and not electric field is detected, these methods are limited to measuring even-order moments of the molecular orientation, regardless of the angle between absorption and emission dipole moments of the molecule.…”