The Rockefeller University Press $30.00 J. Cell Biol. Vol. 193 No. 6 973–983 www.jcb.org/cgi/doi/10.1083/jcb.201101108 JCB 973 JCB: Report Correspondence to Gohta Goshima: goshima@bio.nagoya-u.ac.jp Abbreviations used in this paper: CLASP, CLIP-associated protein; CLIP, cyto- plasmic linker protein; fps, frame per second; mRFP, monomeric RFP; UTR, untranslated region. Introduction Microtubules are dynamic polymers made from /-tubulin dimers and are crucial for various cellular events, such as cell di- vision, polarization, motility, or organelle transport (Desai and Mitchison, 1997). Conventionally, microtubule dynamics cycles are divided into four events: growth, shrinkage, catastrophe (poly- merization to depolymerization transition), and rescue (depoly- merization to polymerization transition; Mitchison and Kirschner, 1984; Horio and Hotani, 1986; Kinoshita et al., 2001). In ad- dition, a “pause” constitutes another state in vivo, where nei- ther rapid polymerization nor depolymerization is observed for certain periods of time (Dhamodharan and Wadsworth, 1995; Desai and Mitchison, 1997; Rogers et al., 2002; Sousa et al., 2007; Yao et al., 2008). In cells, dynamic microtubules are generated with the contribution of nontubulin proteins, particularly those working at the plus ends of microtubules (Howard and Hyman, 2007; Akhmanova and Steinmetz, 2008). The Dis1/XMAP215 family proteins, including yeast Dis1/Stu2, fly Msps (mini spindles), frog XMAP215, and mammalian ch-TOG (colonic and hepatic tumor overexpressed), have tubulin-binding TOG domains, and XMAP215 was shown to promote microtubule growth by processively adding tubulin dimers onto the plus ends and also catalyze the reverse reaction, namely the removal of tubulin from the end, which leads to the promotion of micro- tubule shrinkage (Kerssemakers et al., 2006; Howard and Hyman, 2007; Brouhard et al., 2008; Slep, 2010). Kinesin-13 is a microtubule-depolymerizing kinesin (Desai et al., 1999; Moore and Wordeman, 2004; Rogers et al., 2004a), and its inhibition leads to the suppression of catastrophe and formation of longer microtubules in spindles (Goshima and Vale, 2003; Goshima et al., 2005b; Mennella et al., 2005; Ohi et al., 2007). Interest- ingly, the essential features of physiological microtubule dy- namics were reconstituted by mixing just tubulin, XMAP215, and Kinesin-13 (Kinoshita et al., 2001). Cytoplasmic linker protein (CLIP)–associated proteins (CLASPs; Mast or Orbit in fly) are another class of proteins containing the TOG-like domain and were recently shown to increase rescue and decrease catastro- phe frequency (Al-Bassam et al., 2010). CLIPs (CLIP-190 in fly) promote microtubule growth in some cell types (Brunner and Nurse, 2000; Komarova et al., 2002) but may not in others (Dzhindzhev et al., 2005; Goshima et al., 2007). H ighly conserved EB1 family proteins bind to the growing ends of microtubules, recruit multiple cargo proteins, and are critical for making dynamic microtubules in vivo. However, it is unclear how these master regulators of microtubule plus ends promote microtubule dynamics. In this paper, we identify a novel EB1 cargo protein, Sentin. Sentin depletion in Drosophila mela- nogaster S2 cells, similar to EB1 depletion, resulted in an increase in microtubule pausing and led to the formation of shorter spindles, without displacing EB1 from growing microtubules. We demonstrate that Sentin’s association with EB1 was critical for its plus end localization and function. Furthermore, the EB1 phenotype was rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 is replaced with Sentin. Knockdown of Sentin attenuated plus end accumu- lation of Msps (mini spindles), the orthologue of XMAP215 microtubule polymerase. These results indicate that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin and possibly also a microtubule polymerase to the microtubule tip. EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells Wenjing Li, 1 Tomohiro Miki, 1 Takashi Watanabe, 2 Mai Kakeno, 2 Ikuko Sugiyama, 2 Kozo Kaibuchi, 2 and Gohta Goshima 1 1 Division of Biological Science, Graduate School of Science, and 2 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya 464-8601, Japan © 2011 Li et al. This article is distributed under the terms of an Attribution–Noncommercial– Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). THE JOURNAL OF CELL BIOLOGY