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Vol. 16, Issue 11, 5334-5345, November 2005

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EB1 and EB3 Control CLIP Dissociation from the Ends of Growing Microtubules

Yulia Komarova ^*, Gideon Lansbergen , Niels Galjart , Frank Grosveld , Gary G. Borisy ^*, and Anna Akhmanova 

^* Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611; MGC Department of Cell Biology and Genetics, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

Submitted July 11, 2005; Revised August 19, 2005; Accepted August 30, 2005
Monitoring Editor: Yixian Zheng

EBs and CLIPs are evolutionarily conserved proteins, which associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. In this study we investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. We showed that both CLIPs bind to EB proteins directly. The C-terminal tyrosine residue of EB proteins is important for this interaction. When EB1 and EB3 or all three EBs were significantly depleted using RNA interference, CLIPs accumulated at the MT tips at a reduced level, because CLIP dissociation from the tips was accelerated. Normal CLIP localization was restored by expression of EB1 but not of EB2. An EB1 mutant lacking the C-terminal tail could also fully rescue CLIP dissociation kinetics, but could only partially restore CLIP accumulation at the tips, suggesting that the interaction of CLIPs with the EB tails contributes to CLIP localization. When EB1 was distributed evenly along the microtubules because of overexpression, it slowed down CLIP dissociation but did not abolish its preferential plus-end localization, indicating that CLIPs possess an intrinsic affinity for growing microtubule ends, which is enhanced by an interaction with the EBs.

Abbreviations used: GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; HIS, 6x histidine tag; co-IP, coimmunoprecipitation; mAb, monoclonal antibody; MT, microtubule; RNAi, RNA interference; siRNA, small interfering RNA; +TIPs, plus-end tracking proteins; YFP, yellow fluorescent protein.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Anna Akhmanova (anna.akhmanova{at}chello.nl).

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