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.
|Original language||English (US)|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Jun 6 2017|
Bibliographical noteFunding Information:
We thank Dr. J. H. Lewis for assistance with software development, M. S. Woody for providing insight on angular measurement corrections and programming, and Dr. P. Purohit for helpful discussion regarding torsional stiffness. The MD simulations used in this work were made possible through the Blue Waters sustained-petascale computing project supported by National Science Foundation (NSF) Awards OCI- 0725070 and ACI-1238993, the State of Illinois, and the "Computational Microscope" NSF Petascale Computing Resource Allocations Award ACI-1440026. This work was funded by NIH Grants P01-GM087253, R01-GM086352, and 9P41GM104601, and by NIH Training Grant T32-GM008275.
- Molecular dynamics
- Single molecule