mDia2 stabilizes MTs against depolymerization in vitro In addition to our data, there is already some evidence suggesting there might be a direct role of mDia on MTs. Previous studies have shown that GBDmDia2 localizes along the length of stabi- lized MTs (Palazzo et al., 2001). The related protein, mDia1, is also found at a low frequency at the ends of stable MTs in cells (Wen et al., 2004) and has been localized to the mitotic spindle (Kato et al., 2001). However, to date, there have been no studies to explore whether any member of the mDia family shows direct biochemical activity toward MTs. We tested whether mDia2 might exhibit direct MT binding and stabilizing activity and, if so, whether this activity was sufficient to generate capped MTs in vitro, a defining characteristic of Glu MTs in vivo. We first looked at binding of purified recombinant mDia2 to purified MTs or tubulin dimers. We incubated purified His-, GST-, or untagged versions of FH1FH2mDia2 with taxol- assembled MTs or BSA, followed by high-speed centrifugation to separate the MT pellet from soluble unbound material. Both tagged and untagged mDia2 were substantially recovered in the pellet fraction only when MTs were present (Fig. 4 A). In con- trast, we did not detect any binding of FH1FH2mDia2 with tu- bulin dimer alone by gel filtration, native gel electrophoresis, or Biocore analyses (unpublished data). Binding of FH1FH2mDia2 to MTs was saturable with an estimated stoichiometry at satura- tion of 1:4.7 (mDia2/tubulin) and an estimated KD of 6.1 μM (Fig. 4 B). Similar binding to MTs was observed for the K853A, I704A, and W630A FH1FH2 mutants, although the W630A mutant appeared to bind with higher affinity (Fig. 4, C and D). Shearing MTs to increase the number of MT ends did not de- tectably affect binding of wild-type or mutant FH1FH2mDia2 to MTs (Fig. 4 E). Consistent with the stoichiometry and lack of evidence for preferential end binding, FH1FH2mDia2 was lo- calized along the length of MTs by immunostaining (Fig. 4 F). These results show that FH1FH2mDia2 directly binds to MTs and appears to interact with the sides of the MT. We next tested the effect of mDia2 on MT stability and incu- bated substoichiometric amounts of His- or GST-FH1FH2mDia2 proteins with purified tubulin during spontaneous assembly of MTs at 37°C, followed by a short incubation at 4°C to induce rapid MT depolymerization. SDS gel analyses of MT pellets Figure 5. FH1FH2mDia2 stabilizes MTs against cold-induced depolymerization. (A) SDS gel analysis of the resistance of self-assembled MTs to cold-induced depolymerization after incubating with His- (His-mDia2) or GST- (GST- mDia2) tagged FH1FH2mDia2. After centri- fugation, equivalent amounts of the pellets and supernatants were resolved by SDS-PAGE and visualized by Coomassie. (B) Immunostaining of MTs that survive cold-induced depolymerization in the presence of His-FH1FH2mDia2 (His-mDia2) or buffer alone. (C) SDS gel analysis of the re- sistance of self-assembled MTs to cold-induced depolymerization after incubating with wild-type and mutant His-FH1FH2mDia2. Pellets at 4°C and input material are shown. Panels shown are from a single gel. Bar, 10 μm. showed that addition of His- or GST-tagged FH1FH2mDia2 did not increase the level of MTs pelleting at 37°C but did increase the level of MTs pelleting after cold treatment, which is consistent with a role for mDia2 in MT stabilization (Fig. 5 A). Similar cold stability of MTs was observed when FH1FH2mDia2 was added to preassembled MTs (unpublished data). The stabilizing effect of mDia2 on MTs was concentration dependent and independent of the presence of a tag on mDia2, as untagged FH1FH2mDia2 also stabilized MTs against cold treatment (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200709029/DC1). We con- firmed by immunofluorescence microscopy that a substantial number of MTs resisted cold depolymerization when mDia2 was present (Fig. 5 B) and found that both the actin and dimerization mutants of mDia2 retained this activity (Fig. 5 C). We conclude that FH1FH2mDia2 is sufficient to directly stabilize MTs against cold-induced depolymerization. Glu MTs in extracted cells are characteristically resistant to disassembly upon dilution (Infante et al., 2000). Thus, we tested whether wild-type and mutant versions of FH1FH2mDia2 frag- ments could stabilize MTs assembled from purified tubulin against dilution-induced depolymerization. Controls in which MTs were diluted into BSA, heat-denatured mDia2, or His-tagged GSK3 showed low levels of MTs remaining as assayed by immunofluorescence (Fig. 6 A). In contrast, dilution of MTs into wild-type, actin (I704A, K853A), or dimerization (W630A) mutants of FH1FH2mDia2 all reduced dilution-induced MT depolymerization, as indicated by the increased number and length of MTs remaining after dilution (Fig. 6 A). We also noticed that the MTs persisting in the samples incubated with mDia2 proteins consistently appeared thicker than in the control samples, suggesting that mDia2 may also bundle MTs. We con- firmed that FH1FH2 mDia2 bundled MTs by low-speed sedimen- tation assays of MTs in the presence of wild-type FH1FH2mDia2 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb .200709029/DC1). We next used the MT dilution assay to map the MT- stabilizing activity in FH1FH2mDia2. Recombinant fragments of mDia2 lacking the FH1 and the N terminus of the FH2 (FH2 N), portions of the FH2 (FH1FH2 C), or both (FH2C) were tested for their ability to protect MTs from dilution-induced depolymerization (Fig. 6, A and B). Each of these fragments MDIA2 STABILIZES MICROTUBULES • Bartolini et al. 529