purified axonemal fragments using video-enhanced differential interference contrast (DIC) microscopy. MTs assembled in the presence of His-FH1FH2mDia2 had significantly slower rates of assembly at both plus and minus ends compared with those of MTs assembled from purified tubulin alone (Table I). Although these effects were relatively modest ( 25% inhibition of control rates for both plus and minus ends), these data dem- onstrate that mDia2 inhibits tubulin addition at MT ends. No significant difference was observed in the catastrophe frequen- cies (Cf) or rates of shortening (Vs) during MT assembly in the presence of mDia2. However, because catastrophes and short- ening events are rare under MT assembly conditions, we also explored the effect of FH1FH2mDia2 on MTs under disassembly conditions, where shortening events are predominant and can be analyzed with higher statistical confidence. In these ex- periments, MTs were first assembled on axonemes and then the chamber was perfused with either buffer alone or His- FH1FH2mDia2 diluted in buffer to initiate MT depolymerization. As shown in Fig. 7 A, MTs diluted into His-FH1FH2mDia2 depolymerized more slowly than MTs diluted into buffer alone (Videos 5 and 6, available at http://www.jcb.org/cgi/content/ full/jcb.200709029/DC1). Quantification of this effect showed that in His-FH1FH2mDia, both MT plus and minus ends shortened at 60% of the rate of MTs diluted into buffer alone (Fig. 7 B). The decrease in shortening rate with His-FH- 1FH2mDia2 was concentration dependent and saturated at 2 μM mDia2 (Fig. 7 C). Interestingly, when MTs were induced to depolymerize in the presence of His-FH1FH2mDia2, we occa- sionally observed individual MTs abruptly switching to a slower shortening rate that approached zero (Fig. 7 D). This time-dependent change in the shortening rate occurred only at plus ends. We quantified this behavior by counting shortening ends in which the rate slowed to 50% or less of the starting shortening rate for that MT and found that for 2 μM mDia2, 14.7% of plus ends shifted to a slower shortening rate compared with 0% for the buffer control. Collectively, these data demon- strate that His-FH1FH2mDia2 stabilizes MTs by inhibiting both the polymerization and depolymerization rates at MT ends and suggest that this activity may contribute to the induction of MT stability in vivo. Discussion Formins have been extensively characterized for their ability to nucleate and elongate actin filaments (Goode and Eck, 2007). However, their regulation of the MT cytoskeleton, which has been documented both in dividing and migrating cells, is poorly understood. In NIH3T3 cells, mDia1 is both necessary and suf- ficient to stabilize a subset of MTs (Glu MTs) arranged in the direction of migration downstream from Rho signaling (Palazzo et al., 2001; Goulimari et al., 2005; Eng et al., 2006). mDia also participates in MT stabilization in embryonic endodermal cells and C6 glioma cells (Kodama et al., 2003; Yamana et al., 2006), and evidence exists that mDia associates with MTs and MT ends in vivo (Kato et al., 2001; Palazzo et al., 2001; Wen et al., 2004). However, given the potent and direct effects of mDia on actin assembly, it has remained unclear whether the MT effects Table I. Dynamics of MTs assembled with FH1FH2mDia2 or buffer alone Buffer mDia2 Plus ends Ve = 1.82 ± 0.54 (25) Te = 0.95 ± 0.59 (25) Vs = 30.20 ± 7.90 (11) Ts = 0.21 ± 0.09 (11) Cf = 0.44 ± 0.13 (11) Ve = 1.42 ± 0.47 (30)** Te = 0.97 ± 0.62 (30) Vs = 35.57 ± 14.78 (8) Ts = 0.15 ± 0.07 (8) Cf = 0.27 ± 0.09 (8) Minus ends Ve = 0.99 ± 0.23 (21) Te = 0.96 ± 0.57 (21) Vs = 27.67 ± 11.76 (3) Ts = 0.17 ± 0.06 (3) Cf = 0.14 ± 0.08 (3) Ve = 0.75 ± 0.45 (40)* Te = 1 ± 0.58 (40) Vs = 28.73 ± 26.07 (6) Ts = 0.25 ± 0.22 (6) Cf = 0.14 ± 0.05 (6) Dynamics obtained with axonemal fragments using 10 μM PC tubulin and 2 μM His-FH1FH2mDia2 (mDia2) or buffer alone. Values are mean ± SD with the num- ber of MTs observed indicated in parentheses. Ve, elongation rate (μm/min); Te, time spent in elongation (min); Vs, shortening rate (μm/min); Ts, time spent in shortening (min); Cf, catastrophe frequency (min-1). *, P < 0.05; **, P < 0.01 (Student’s t test). might occur as a secondary consequence of mDia-induced actin assembly in cells. In this paper, we show that promotion of actin polymerization and induction of stable Glu MTs by mDia2 can be functionally separated, as two actin polymerization-defective mutants of mDia2 were capable of inducing stable Glu MTs in NIH3T3 cells (Figs. 1 and 2). We also demonstrate that a sta- ble mDia2 dimer is not required to generate stable Glu MTs (Figs. 3 and 6). As the stable dimer is thought to be the func- tional state of formins during actin assembly (Copeland et al., 2004; Moseley et al., 2004; Xu et al., 2004), this finding rein- forces the notion that processive barbed end actin assembly and induction of MT stability are functionally separable. Interest- ingly, the ablation of actin assembly or processive barbed end actin filament association by mDia2 mutants was consistently coupled to induction of stable Glu MTs that appeared knot- ted around the centrosome instead of being fully extended to the cell periphery (Figs. 2, 3, and 6). We have not analyzed the cause of this phenotype, but as cells that express mutant mDia2 showed normal cell morphology and had a normal distribu- tion of Tyr MTs, it appears that the perinuclear localization of Glu MTs was not a result of cell contraction or global effects on MTs. Rather, this phenotype suggests that the ability of mDia2 to function on the actin cytoskeleton may still be re- quired for proper distribution of stable MTs, specifically to ori- ent stable MTs toward the leading edge. Perhaps mDia’s ability to interact with dynamic actin filaments at this cellular site creates a localized pool of mDia that can also interact with nearby MTs. Interestingly, integrin engagement and cluster- ing into focal adhesions, which is partly dependent on mDia (Riveline et al., 2001; Yamana et al., 2006), is also necessary for formation of stable Glu MTs oriented toward the leading edge (Palazzo et al., 2004). The finding that actin remodeling by mDia2 was not re- quired for stabilizing MTs in cells prompted us to test whether formins could affect MT stability directly. Indeed, we observed that an FH1FH2 fragment of mDia2 stabilized MTs assembled from purified tubulin against depolymerization induced by ei- ther cold or tubulin dilution (Figs. 4, 6, and 7). As induction of MDIA2 STABILIZES MICROTUBULES • Bartolini et al. 531