Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends |
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Authors: | Dragestein Katharina A van Cappellen Wiggert A van Haren Jeffrey Tsibidis George D Akhmanova Anna Knoch Tobias A Grosveld Frank Galjart Niels |
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Institution: | 1Department of Cell Biology and Genetics and 2Department of Reproduction and Development, Erasmus Medical Center, 3000 DR Rotterdam, Netherlands;3Institute of Electronic Structure and Laser, Foundation for Research and Technology - Hellas, 71110 Heraklion, Crete, Greece |
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Abstract: | Microtubule (MT) plus end-tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end. |
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