Insights into the process of EB1-dependent tip-tracking of kinesin-14 Ncd. The role of the microtubule
| Autor: | Ewa Szczęsna, Andrzej A. Kasprzak |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Histology Total internal reflection fluorescence microscope fungi Mutant Kinesins macromolecular substances Cell Biology General Medicine Biology Microtubules Pathology and Forensic Medicine Microtubule plus-end Motor protein 03 medical and health sciences Dwell time Drosophila melanogaster 030104 developmental biology Microtubule Biophysics Animals Drosophila Proteins Kinesin Microtubule-Associated Proteins Process (anatomy) |
| Zdroj: | European Journal of Cell Biology. 95:521-530 |
| ISSN: | 0171-9335 |
| Popis: | End-binding proteins are capable of tracking the plus-ends of growing microtubules (MTs). The motor protein Ncd, a member of the kinesin-14 family, interacts with EB1 protein and becomes a non-autonomous tip-tracker. Here, we attempted to find out whether at least for Ncd, the efficient EB1-mediated tip-tracking involves the interaction of the kinesin with the MT surface. We prepared a series of Ncd tail mutants in which the MT-binding sites were altered or eliminated. Using TIRF microscopy, we characterized their behavior as tip-trackers and measured the dwell times of single molecules of EB1 and Ncd tail or its mutated forms. The mutated forms of Ncd tail exhibited tip-tracking in the presence of EB1 and the effectiveness of this process was proportional to the affinity of the mutant's tail to MT. Even though the interaction of Ncd with EB1 was weak (Kd∼9μM) the half saturating concentration of EB1 for tip-tracking was 7nM. The dwell time of Ncd tail in the presence of EB1 was ∼1s. The dwell time of EB1 alone was shorter (∼0.3s) and increased considerably in the presence of a large excess of Ncd tail. We demonstrated that tip-tracking of kinesin-14 occurs through several concurrent mechanisms: binding of kinesin only to EB1 located at the MT end, interaction of the kinesin molecules with a composite site formed by EB1 and the MT tip, and probably surface diffusion of the tail along MT. The second mechanism seems to play a crucial role in efficient tip-tracking. |
| Databáze: | OpenAIRE |
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