Kinesin-8 motors regulate the lengths of microtubules in cells. In previous studies, these motors have been shown to utilize their highly processive plus-end directed motility to reach microtubule plus-ends where they act as a microtubule depolymerase. The superprocessive motility importantly allows Kip3 motors to depolymerize microtubules in a length-dependent manner, the underlying mechanism of which has been described by an antenna model. During such long runs, motors in vivo are expected to frequently encounter roadblocks, such as microtubule associated proteins. The adaptions in the stepping mechanism that allow kinesin-8 motors to navigate around roadblocks to reach microtubule ends is not well understood. In this work, in vitro techniques were utilized to understand the navigation strategy of yeast kinesin-8, Kip3.
Three-dimensional stepping motility of Kip3 on the surface of microtubules can be inferred (i) indirectly from rotational motion of microtubules gliding along a surface coated with Kip3 and (ii) directly by three-dimensional tracking of Kip3 on freely suspended microtubules. Firstly, an impact-free method to detect rotations of gliding microtubules was established based on fluorescent speckles within the microtubule structure in combination with fluorescent interference contrast microscopy. Secondly, a suspended microtubule assay was established to obtain three- dimensional trajectories of single Kip3 motors, using Parallax, a dual-focus imaging technique.
The motility assays performed in this work revealed that Kip3 motors undergo left-handed helical motion around the microtubule lattice. This indicates that Kip3 employs a directed sidestepping strategy which is attributed to the motor having a flexible neck and/or a long neck linker. Interestingly, further analysis of the rotational motion revealed that the sidestepping of Kip3 is not directly coupled to the forward stepping. Based on these observations, it is hypothesized that the motor can transition from a two-head-bound conformation to a one-head-bound conformation while waiting for ATP. Whereas the motor can step forward from both states, sidestepping is strongly favored from the one-head-bound conformation. This hypothesis was confirmed through experiments as well as numerical simulations where the transition from the two-head-bound conformation to the one-head-bound conformation was enhanced by either prolonging the ATP waiting time or increasing the transition rate (by reducing the motor-microtubule interaction).
Finally, Kip3 based motility assays were performed using microtubules decorated with rigor binding kinesin-1 motors acting as roadblocks. While gliding assays using roadblock-decorated microtubules indicated a left-biased sidestepping strategy for Kip3, stepping assays revealed an additional diffusive component in the stepping motility of Kip3, along with the leftward bias. Taken together, it is hypothesized that Kip3 has a dual-mode roadblock circumnavigation strategy. Upon encountering a roadblock, the motor circumnavigates it (i) by shifting to the adjacent left microtubule protofilament using the biased sidestepping mechanism or (ii) by shifting microtubule protofilaments in an unbiased diffusive manner upon switching out of the step cycle. Therefore, the biophysical properties of Kip3 are fine-tuned to ensure that the motor reaches the microtubule plus-end to perform its depolymerase activity.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:30820 |
| Date | 18 December 2017 |
| Creators | Mitra, Aniruddha |
| Contributors | Diez, Stefan, Schlierf, Michael, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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