T cell proliferation and migration (Eisenmann et al., 2007; Goode and Eck, 2007; Gupton et al., 2007; Sakata et al., 2007). Two members of the Dia family in mouse (mDia1 and mDia2) also regulate endocytosis and endosome trafficking, presumably by promoting endosome actin coat formation and dynamics (Tominaga et al., 2000; Gasman et al., 2003; Fernandez-Borja et al., 2005; Wallar et al., 2007). There is growing evidence that formins may also be involved in regulating MT-dependent processes such as meiotic spindle alignment and chromosome congression at mitosis (Leader et al., 2002; Yasuda et al., 2004). Notably, both mDia1 and its interacting protein Pkd2 localize to mitotic spindles (Ishizaki et al., 2001; Kato et al., 2001; Rundle et al., 2004). In Drosophila melanogaster, the formin Cappuccino directly regulates Rho-dependent actin-MT cross talk during ooplasmic streaming and has an MT binding domain (Rosales-Nieves et al., 2006). In migrating fibroblasts, mDia1 stabilizes a subset of MTs downstream of lysophosphatidic acid (LPA)–induced Rho signaling (Palazzo et al., 2001; Wen et al., 2004; Goulimari et al., 2005; Eng et al., 2006). mDia may also contribute to MTOC orientation in fibroblasts and cytotoxic lymphocytes (Yamana et al., 2006; Gomez et al., 2007). The current model for mDia-regulated stabilization of MTs suggests that LPA triggers a Rho/mDia-dependent pathway that also involves EB1, APC, GSK3 , and novel PKCs to generate a polarized array of stabilized MTs oriented toward the leading edge of cells in a wounded monolayer (Palazzo et al., 2001; Wen et al., 2004; Eng et al., 2006). The localization of these stabilized MTs is regulated by integrin signaling (Palazzo et al., 2004), and this is thought to contribute to cell polarity by directing vesicular trafficking and/or actin regulators to the leading edge (Lin et al., 2002). Rho/mDia-stabilized MTs are characterized by elevated levels of posttranslationally detyrosinated -tubulin, which arises after stabilization through the removal of the C-terminal tyrosine residue (Gundersen et al., 1984, 1987). Detyrosination exposes a glutamate residue at the C terminus of the -tubulin and, hence, stable MTs that contain elevated detyrosinated -tubulin are re- ferred to as Glu MTs, as opposed to more dynamic MTs, which contain tyrosinated -tubulin and are referred to as Tyr MTs (Gundersen et al., 1984, 1987). Analyses of MT plus-end turnover in cells have shown that the stabilization induced by LPA/Rho-mDia signaling is achieved by capping of MT plus ends, which makes them refractory to tubulin subunit exchange (Infante et al., 2000; Palazzo et al., 2001). The biochemical mechanisms of MT stabilization by Rho/mDia proteins are still poorly understood and it is unclear which proteins in the pathway are directly involved in stabilizing this class of MTs. To date, there is no evidence to support a role for formins in directly modulating the dynamics of the MT cyto- skeleton, and it remains possible that MT stabilization is a secondary consequence of mDia-mediated assembly and stabili- zation of actin filaments. In this paper, we show that actin nucle- ation and MT stabilization by mDia2 are two separate cellular functions of this formin and, further, that the formation of a sta- ble mDia2 dimer is not necessary for MT stabilization. We also demonstrate that purified mDia2 directly stabilizes MTs by in- hibiting both polymerization and depolymerization rates and that this activity maps to the FH2 domain. 524 JCB • VOLUME 181 • NUMBER 3 • 2008 Results I704A and K853A FH1FH2mDia2 mutants are defective in actin assembly in vitro and in vivo Alanine substitutions of Ile 1431 and Lys 1601 completely abolish the actin-nucleating activity of the FH2 domain in the yeast formin Bni1p (Xu et al., 2004) and, at least in the case of the corresponding I704A mutation, in mammalian mDia2 (Harris et al., 2006). These substitutions also eliminate the ability of the FH2 domain to protect barbed ends from heterodimeric capping protein (Xu et al., 2004; Harris et al., 2006). We intro- duced the analogous mutations (I704A and K853A) in a consti- tutively active fragment of mDia2 that consists of both the FH1 and 2 domains and lacks the regulatory GBD and DAD domains (Fig. 1 A). We prepared the mutants in this fragment of mDia2 for three reasons: it is the minimal fragment of the protein that is fully active for MT stabilization in vivo (Wen et al., 2004); it is expected to retain its actin nucleation activity because it has intact FH1 and 2 domains; and it does not dimerize with mDia1 (Copeland et al., 2004). We first prepared recombinant His-tagged proteins and tested them for actin assembly using a kinetic assay using pyrene- labeled actin (Fig. 1 B; Li and Higgs, 2003, 2005; Harris et al., 2006). Both mutants were reduced in stimulating actin assembly compared with their wild-type counterpart, with a >1,000-fold reduction in activity for the I704A mutant and 60–70-fold re- duced activity for the K853A mutant. Thus, although the K853A mutation is intermediate in effect, the I704A is virtually dead for actin assembly. To test whether the actin polymerization defect in vitro correlated with a loss of activity in vivo, we introduced wild- type and mutant forms of FH1FH2mDia2 into serum-starved NIH3T3 cells, which have low levels of assembled actin. We injected the proteins at the minimal concentration at which the wild-type fragment induced actin fibers as assessed by phalloidin staining. Under these conditions both mutants failed to induce actin fibers (Fig. 1, C and D). No dominant-negative effects of the mutant constructs were observed in cells that had been stimulated with LPA to induce stress fibers (unpub- lished data). We used total internal reflection fluorescence (TIRF) microscopy to examine the directional movement of mDia2 puncta, which are thought to reflect actin filament growth (Higashida et al., 2004). Consistent with the inability of the mutants to induce actin filaments, we observed fast directional movement of EGFP-FH1FH2mDia2 puncta only with the wild- type construct, as we could not trace linear tracks for most of the EGFP puncta in cells with either of the mDia2 mutants (Fig. 1 E). As previously observed for an analogous mDia1 frag- ment (Higashida et al., 2004), wild-type EGFP-FH1FH2mDia2 puncta typically moved toward the cell periphery and accumu- lated at the tips of filopodia-like protrusions (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200709029/DC1). We confirmed that this movement was dependent on intact actin filaments, but not MTs, because it was blocked by latrunculin A and not by nocodazole (unpublished data). The velocity of