In vitro reconstitution of lateral to end-on conversion of kinetochore-microtubule attachments.
| Autor: | Chakraborty M; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States., Tarasovetc EV; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States., Grishchuk EL; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: gekate@pennmedicine.upenn.edu. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in cell biology [Methods Cell Biol] 2018; Vol. 144, pp. 307-327. Date of Electronic Publication: 2018 May 11. |
| DOI: | 10.1016/bs.mcb.2018.03.018 |
| Abstrakt: | During mitosis, kinetochores often bind to the walls of spindle microtubules, but these lateral interactions are then converted into a different binding mode in which microtubule plus-ends are embedded at kinetochores, forming dynamic "end-on" attachments. This remarkable configuration allows continuous addition or loss of tubulin subunits from the kinetochore-bound microtubule ends, concomitant with movement of the chromosomes. Here, we describe novel experimental assays for investigating this phenomenon using a well-defined in vitro reconstitution system visualized by fluorescence microscopy. Our assays take advantage of the kinetochore kinesin CENP-E, which assists in microtubule end conversion in vertebrate cells. In the experimental setup, CENP-E is conjugated to coverslip-immobilized microbeads coated with selected kinetochore components, creating conditions suitable for microtubule gliding and formation of either static or dynamic end-on microtubule attachment. This system makes it possible to analyze, in a systematic and rigorous manner, the molecular friction generated by the microtubule wall-binding proteins during lateral transport, as well as the ability of these proteins to establish and maintain association with microtubule plus-end, providing unique insights into the specific activities of various kinetochore components. (© 2018 Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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