Microtubule length control is indispensable for cytoskeletal functions such as mitotic spindle assembly and positioning. In vivo studies have shown that kinesin motor proteins can regulate microtubule length positively and negatively. The mechanisms by which kinesins act as depolymerases and catastrophe factors are well studied. By contrast, how kinesins promote microtubule growth is unknown. The aim of this work was to elucidate the mechanism by which budding yeast kinesin Kip2 regulates microtubule dynamics, using in vitro reconstitution assays combined with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy. Kip2 was shown to increase the mean length of microtubules through length-dependent polymerase and anti-catastrophe activities, both with porcine and yeast tubulin, in the absence of accessory proteins. Using single-molecule motility assays, Kip2 was shown to translocate in a highly processive, ATP-dependent manner and to processively target tubulin oligomers to microtubule plus-ends. Mutant studies to probe Kip2 structure-function relationships revealed that the N-terminus of Kip2 is dispensable for promotion of microtubule growth, while the C-terminus is not. An effort to functionally identify a tubulin/microtubule-binding domain in the Cterminus of Kip2 remained unfruitful. Finally, the combinatorial effect of Kip2 with interaction partners Bim1 and Bik1 on microtubule dynamics was reconstituted. This microtubule plus-end tracking complex promoted microtubule growth beyond the effect of Kip2 alone. Together, this work demonstrates that a kinesin motor can act directly as a length-dependent microtubule polymerase and anti-catastrophe factor in the absence of accessory proteins. Thereby, this work provides insight into how kinesins control microtubule length.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:73767 |
| Date | 08 February 2021 |
| Creators | Hibbel, Anneke |
| Contributors | Diez, Stefan, Howard, Jonathon, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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