JCB: ARTICLE Y G O L O I B L L E C F O L A N R U O J E H T The formin mDia2 stabilizes microtubules independently of its actin nucleation activity Francesca Bartolini,1 James B. Moseley,2 Jan Schmoranzer,1 Lynne Cassimeris,3 Bruce L. Goode,2 and Gregg G. Gundersen1 1Department of Pathology, Anatomy and Cell Biology, Columbia University, New York, NY 10032 2Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454 3Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015 A migration is the Rho/mDia-dependent stabiliza- critical microtubule (MT) polarization event in cell tion of a subset of MTs oriented toward the direc- tion of migration. Although mDia nucleates actin ﬁlaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mu- tants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization- impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution- induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, re- spectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nu- cleation activity. Introduction Formins are a family of ubiquitous, conserved proteins that are implicated in a wide range of actin-based processes, such as cell polarization, cytokinesis, and cell morphogenesis. Formins are defined by their signature formin homology (FH) 2 domain, which assembles unbranched actin structures from the barbed ends (Pruyne et al., 2002; Sagot et al., 2002). The FH2 domain forms a tethered dimer consisting of two antiparallel elongated actin binding domains (Shimada et al., 2004; Xu et al., 2004). Mutation of conserved tryptophans at the dimerization interface abrogates actin assembly activity, suggesting that the dimer is the functional state of formin proteins (Moseley et al., 2004; Xu et al., 2004). Adjacent to the FH2 domain is a proline-rich FH1 domain that can accelerate the rate of filament elonga- tion through interactions with profilin-bound actin monomers (Sagot et al., 2002; Kovar et al., 2003, 2006; Li and Higgs, 2003; Pring et al., 2003; Moseley et al., 2004; Romero et al., 2004; Vavylonis et al., 2006). Correspondence to Gregg G. Gundersen: ggg1@columbia.edu J.B. Moseley’s present address is Laboratory of Yeast Genetics and Cell Biology, The Rockefeller University, New York, NY 10021. Abbreviations used in this paper: DAD, diaphanous autoregulatory domain; DIC, differential interference contrast; DRF, Diaphanous-related formin; FH, formin homology; GBD, GTPase binding domain; LPA, lysophosphatidic acid; MT, microtubule; TIRF, total internal reﬂection ﬂuorescence. The online version of this paper contains supplemental material. © 2008 Bartolini et al. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 181 No. 3 523–536 www.jcb.org/cgi/doi/10.1083/jcb.200709029 Diaphanous-related formins (DRFs) include Dia (diapha- nous), DAAM (disheveled-associated activators of morphogen- esis), and FRL (formin-related proteins identified in leucocytes) in mammals and Bni1 and Bnr1 in yeast (Higgs and Peterson, 2005; Rivero et al., 2005). The DRF subfamily is characterized by domains that regulate the subcellular localization and formin activation by Rho family GTPases. The binding of Rho-GTP to the GTPase binding domain (GBD) is thought to activate DRFs by relieving an autoinhibitory interaction between the adjacent N-terminal diaphanous inhibitory domain and a short segment located on the C-terminal side of the FH2 domain, the diapha- nous autoregulatory domain (DAD; Watanabe et al., 1999; Alberts, 2001; Li and Higgs, 2005; Otomo et al., 2005; Rose et al., 2005). Functional consequences of DRF activation generally depend on the ability of DRFs to nucleate actin polymerization, resulting in the formation of linear actin filaments such as actin cables in yeast or actin fibers and filopodia in higher eukaryotes. Cellular functions mediated by DRFs include both cytoskeletal rearrangements and transcriptional activation of specific pro- moters such as the serum response element (Copeland and Treisman, 2002). DRF’s ability to remodel the actin cytoskeleton has been implicated in cytokinesis, tissue morphogenesis, cell polarity, and cell adhesion, where they appear to be required for filopodia formation, focal adhesion turnover, and, more recently, JCB 523