in vivo. This may be accomplished by the formation of a multi- protein complex at MT plus ends in which all the players con- tribute to the stabilization activity. Because we have localized FH1FH2mDia2 predominantly to the sides of MTs (Fig. 4, E and F), it will be important to determine whether these additional factors restrict the activity of mDia2 toward MT ends or whether side binding to MTs by mDia2 contributes to the recruitment of other stabilizing factors to the plus end. We attempted to narrow down the MT stabilization activ- ity on mDia2 but found that there are at least two domains within the FHIFH2 fragment. We could not purify a His-tagged FH1 fragment of mDia2, yet no MT binding was detected for in vitro–translated FH1 fragment (unpublished data). These data suggest that the FH2 fragment contains at least two MT stabili- zation sites: one encompassing residues 616–910 and the other encompassing residues 910–1040. Each site is sufficient to in- duce MT stability in vitro and in vivo. The presence of two sep- arate MT binding sites is also consistent with stabilization induced by a dimerization-defective W603A mutant of mDia2 and may explain the apparent bundling of MTs observed in the dilution assays (Figs. 3, 6, and S2). Bundling of MTs with actin filaments has previously been observed for Cappuccino, a re- lated formin in D. melanogaster (Rosales-Nieves et al., 2006). Although bundling of MTs with themselves or with actin fila- ments could contribute to MT stability, we note that it cannot explain all of mDia2’s stabilizing activity toward MTs be- cause mDia2 also enhanced stability of individual MTs in the axonemal assays. Our results show that the MT stabilization domain of mDia2 maps within the same region that is involved in actin polymerization (FH2 core domain). This finding raises the pos- sibility that there is competition between the two cytoskeletal systems for mDia2. Importantly, the activity of mDia2 for actin is in the nanomolar range, whereas that for MTs is in the micro- molar range. A simple competition model would predict that if actin and MTs exhibit mutually exclusive for binding to mDia, then redistribution of the formin to the MT cytoskeleton can only occur if the affinity for MTs is significantly increased. Potentially, this may be mediated by any of several regulatory proteins already implicated in stable Glu MT formation down- stream of LPA signaling, including the MT TIP proteins APC and EB1 (Wen et al., 2004) and the kinases novel PKC and GSK3 (Eng et al., 2006). Selective activation of these mDia partners could localize mDia to MTs by preferential association of the complexes to MTs or by blocking high-affinity actin binding sites on the formin itself. FH1FH2mDia2 fragments can stabilize MTs directly by inhibiting MT dynamics (Figs. 4, 6, and 7) and the stabilization activity maps to the FH2 domain of mDia2, the most conserved region among DRFs. These observations indicate that induction of MT stability is likely to be a shared feature of many DRFs. To date, only mDia1 has been shown to be necessary for stable MT formation in migrating cells. It will be important to test whether other DRFs can stabilize MTs directly and whether this activity is required in other cellular processes in which DRFs have already been implicated, such as cell adhesion, mitosis, or organelle trafficking. Materials and methods Chemicals All chemicals were obtained from Sigma-Aldrich unless otherwise noted. Cell culture, microinjection, and transfection NIH3T3 cells were grown in DME and 10% calf serum as previously de- scribed (Cook et al., 1998; Kreitzer et al., 1999; Palazzo et al., 2001). For microinjection, conﬂuent NIH3T3 cells grown on acid-washed cover- slips were serum starved for 48 h in DME plus 10 mM Hepes, pH.7.4, and wounded for 30 min before being microinjected as previously described (Cook et al., 1998; Kreitzer et al., 1999; Palazzo et al., 2001). DNA plasmids in HKCL buffer (10 mM Hepes, pH.7.4, and 140 mM KCL) at 50–200 μg/ml were microinjected into nuclei and allowed to express for 2 h. Puriﬁed proteins were microinjected into the cytoplasm in HKCL buffer at 2 mg/ml, unless otherwise indicated, and incubated for 1 h and 30 min before ﬁxation. In the experiments in which His-FH1FH2mDia2 proteins were used, 1 mg/ml of the fusion protein was coinjected with 5 mg/ml of the injection marker human IgG. NIH3T3 cells were transfected in growth medium (DME with 10% calf serum) with Lipofectamine Plus (Invitrogen), according to the manufacturer’s speciﬁcations, and allowed to express for an additional 18–24 h. Plasmid construction pGST APC-C (aa 2167 2843) and GST-FH1FH2mDia2 (aa 521–1040) were gifts from R. Baer (Columbia University, New York, NY) and A. Alberts (Van Andel Institute, Grand Rapids, MI), respectively. pGST-EB1 and pHis-FH1FH2mDia2 were constructed as described previously (Wen et al., 2004). pEGFP-FH1FH2mDia2 was prepared by subcloning 521–1040- mDia2 from pEFm-EGFP (Wen et al., 2004) into pEGFP-C1 (Clontech Lab- oratories, Inc.). K853A, I704A, and W630A mDia2 point mutants were generated on pGST-FH1FH2mDia2 using the QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s protocol and using the following synthetic sense oligonucleotide primers: 5 -ttgct- cagaacctttcagccttcctgagctccttccg-3 (I704A); 5 -ctctgtaaactgaaggatacag- catctgcagatcagaaaaccaca-3 (K853A); and 5 -gaaatcagcatgagaagatt- gaatgcgttaaagatcggaccaaatgaaat-3 (W630A). All mutations were sub- cloned into pEGFP-C1 and pQ30E by cutting the inserts with BamH1 and Xho1 and the vectors with BamH1–Xho1 or BamH1–Sal1, respectively. His-FH1FH2mDia2 deletion constructs were all generated by PCR ampliﬁ- cation of pQ30E-FH1FH2mDia2 using KOD HiFi polymerase (EMD) and the following sense oligonucleotide primers: 5 -gcggatccgcagagttacaagctt-3 (His-FH1FH2 C); 5 -gcggatccgctgtcagtactg-3 (His-FH2 N); and 5 -gcg- gatccgagaagaatttggaa-3 (His-FH2C). 5 -gcggtaccctgttgaagctgccttc-3 (His- FH1FH2 C) and 5 -gcggtacccactcctgtctcatctc-3 (His-FH2 N and His-FH2C) were chosen as the antisense oligonucleotide primers. pEGFP-FH1FH2 C and pEGFP-FH2C were constructed by subcloning the inserts from pQ30E into pEGFP-C1 using BamH1–Kpn1 and Bgl2–Kpn1 to cut inserts and vector, respectively. All constructs were veriﬁed by sequencing, and DNA was puri- ﬁed with midiprep kits (QIAGEN). Protein puriﬁcation and GST pulldown assays GST-tagged proteins (GST-APC-C aa 2167 2843, GST-EB1, and wild- type GST-FH1FH2mDia2) and wild-type and mutant His-FH1FH2mDia2 were expressed in E. coli BL-21 or XL-1 blue cells and afﬁnity puriﬁed on agarose-coupled glutathione (GE Healthcare) or nickel resin (Clontech Lab- oratories, Inc.), respectively, according to the manufacturer’s recommended protocols. Untagged FH1FH2mDia2 was obtained by thrombin-mediated cleavage of the GST from the GST fusion protein according to the manufac- turer’s protocol. Puriﬁed proteins were resuspended in HKCL buffer plus 1 mM DTT using Sephadex G-25 PD-10 desalting columns (GE Healthcare). Direct binding was performed by incubating 0.3 μM His-FH1FH2mDia2 with 0.3 μM GST EB1 or GST APC-C in modiﬁed RIPA buffer (PBS, 1% Nonidet-P40, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) for 18 h at 18°C as previously described (Wen et al., 2004). Bound proteins were analyzed by Western blotting with mouse RGS-His antibody (1:10,000). Gel ﬁltration assays Afﬁnity puriﬁed His-FH1FH2mDia2 proteins were analyzed by size exclu- sion chromatography using a Superdex 200 HR 10/30 column (GE Healthcare) and a Controller LCC-501 Plus FPLC system (GE Healthcare). The column was preequilibrated and run in HKCL buffer plus 1 mM DTT. The elution was monitored by absorbance at 280 nm and veriﬁed by SDS- PAGE of column fractions followed by Western blotting using mouse RGS-His MDIA2 STABILIZES MICROTUBULES • Bartolini et al. 533