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Published online
doi:10.1083/jcb.200707203
The Journal of Cell Biology, Vol. 180, No. 4, 729-737
The Rockefeller University Press, 0021-9525 $30.00
© Dragestein et al.
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Dynamic behavior of GFP–CLIP-170 reveals fast protein turnover on microtubule plus ends



Katharina A. Dragestein1, Wiggert A. van Cappellen2, Jeffrey van Haren1, George D. Tsibidis3, Anna Akhmanova1, Tobias A. Knoch1, Frank Grosveld1, and Niels Galjart1

1 Department of Cell Biology and Genetics and 2 Department of Reproduction and Development, Erasmus Medical Center, 3000 DR Rotterdam, Netherlands
3 Institute of Electronic Structure and Laser, Foundation for Research and Technology - Hellas, 71110 Heraklion, Crete, Greece

Correspondence to Niels Galjart: n.galjart{at}erasmusmc.nl

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.

Abbreviations used in this paper: CLIP-170, cytoplasmic linker protein 170; EB, end-binding protein; FCA, fluorescence cumulant analysis; FCS, fluorescence correlation spectroscopy; MEF, mouse embryonic fibroblast; MT, microtubule; PCH, photon-counting histogram; ROI, region of interest.


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