Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends.

Microtubule (MT) plus end – tracking proteins (+TIPs) specifi cally 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 specifi city 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 affi nity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fl uorescence-based approaches, we show that two +TIPs, CLIP-170 and endbinding 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 affi nity. We propose that the observed loss of fl uorescent +TIPs at plus ends does not refl ect the behavior of single molecules but is a result of overall structural changes of the MT end.

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