Motor dynamics underling bidirectional cargo transport by kinesin and dynein
Open Access
- Author:
- Feng, Qingzhou
- Graduate Program:
- Molecular, Cellular and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 30, 2020
- Committee Members:
- William O Hancock, Dissertation Advisor/Co-Advisor
Melissa Rolls, Committee Member
Lu Bai, Committee Member
Charles T Anderson, Committee Member
Christopher Martin Yengo, Outside Member
Melissa Rolls, Program Head/Chair
William O. Hancock, Committee Chair/Co-Chair - Keywords:
- kinesin
dynein
microtubule
bidirectional transport - Abstract:
- Microtubule-based cargo transport by kinesin and dynein motor proteins is essential for cellular function, particularly in neurons due to their extended axons and dendrites. Thanks to the development of single molecule tracking technology, single motor protein properties are well-studied across the kinesin family. Similarly, recent work on activated dynein complexes has provided important insights into the properties of dynein motors. However, most cargo in cells are transported by multiple motors, and many cellular observations cannot be simply explained based on the properties of individual motors. This work focuses on the cooperative and antagonistic motor interactions that underlie bidirectional cargo transport in cells. The first study investigated multi-motor transport by kinesins. Kinesin-based cargo transport frequently involves the coordinated activity of kinesins from different families that move at different speeds. To understand these multimotor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be four-fold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motors with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than does kinesin-1. Finally, compared to cargo transported by two kinesin-1, cargo transported by two kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads whereas kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules. The second study investigated the mechanism of dynein activation and the competition between pairs of kinesin and dynein. Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used Interferometric Scattering (iSCAT) microscopy to track dynein-dynactin-BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive and stuck motility states. We found that DDB spends 65% of its time undergoing processive stepping, 4% undergoing one-dimensional diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 surprisingly enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB - kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity of the complexes, in quantitative agreement with the shift of isolated DDB into the diffusive state. The data suggest a DDB activation model in which dynactin p150 enhances dynein processivity not solely by acting as diffusive tether that maintains microtubule association, but rather by acting as an allosteric activator that promotes a conformation of dynein optimal for processive stepping. Together, these studies support a model in which kinesin functional diversity and dynein activation play important roles in regulating the bidirectional transport, and suggest that motors can act as tethers to enhance microtubule interactions during transport and that motors are mechanically activated by their antagonistic partners through mechanisms that are still being uncovered.