Figure 6. FH1FH2mDia2 stabilizes MTs against dilution-induced disassembly and the activity maps to the FH2 domain. (A) Immuno- staining of MTs that survived dilution induced depolymerization upon incubation with the fol- lowing indicated proteins (all proteins were His- tagged except BSA): GSK3 , glycogen synthase kinase 3 ; WT, wild-type FH1FH2mDia2; I704A, K853A, and W630A, mutant FH1FH2mDia2; FH1FH2 C, FH2 N, and FH2C, fragments of FH1FH2mDia2 (see B). (B) Diagram of fragments of FH1FH2mDia2 used for MT stability tests in A. (C) Immunostaining of Glu and Tyr MTs and actin in wounded starved NIH3T3 ﬁbroblasts expressing EGFP-FH1FH2 C or EGFP-FH2C- mDia2. (D) Quantiﬁcation of cells expressing the indicated constructs that exhibited stable Glu MTs. Data are mean ± SD from three inde- pendent experiments (n > 70 cells). Asterisk indicates P < 0.001, calculated by 2 test (one degree of freedom). Bars, 10 μm. stabilized MTs against dilution-induced depolymerization (Fig. 6 A). Collectively, these results suggest that at least two MT stabilization sites are present in FH1FH2mDia2: one en- compasses residues 521–910 (FH1FH2 C) and the other resi- dues 910–1040 (FH2C). We next tested whether the FH1FH2 C and FH2C frag- ments induced stable MTs in serum-starved cells and found that both induced stable Glu MTs above background levels (Fig. 6, C and D). Interestingly, as previously noted for the mDia2 actin and dimerization mutants, these Glu MTs appeared knotted and clustered at a perinuclear region rather than extending to the cell periphery as observed for stable Glu MTs induced by LPA. To examine whether this knotted phenotype reflected a loss in actin nucleation activity, we tested these fragments for their ability to assemble F-actin. Neither deletion construct induced formation of actin filaments comparable to FH1FH2mDia2 levels in serum- starved cells (Fig. 6 C). The FH2C fragment also showed almost a 1000-fold lower activity than FH1FH2mDia2 in pyrene-actin assembly assays (Fig. S3, available at http://www.jcb.org/cgi/ content/full/jcb.200709029/DC1). In addition, no linear tra- jectories of EGFP particles were observed when EGFP-tagged versions of these fragments were transfected into NIH3T3 cells and analyzed by TIRF microscopy (unpublished data). These results were not surprising, as an intact FH2 dimer is required for productive interactions of formins with actin (Moseley et al., 2004; Xu et al., 2004). The loss of actin assembly activity in these fragments of mDia2 that maintain MT stabilizing activ- ities confirms a clear separation between mDia2 activities on actin and MTs. mDia2 decreases tubulin dissociation rates in vitro MT stabilization by the Rho–mDia pathway occurs by inhibition of tubulin loss and gain at MT ends (Palazzo et al., 2001). The stability assays employed in the experiments shown in Figs. 4, 5, and 6 do not reveal whether the MT stabilization activity of mDia2 is mediated by an inhibitory effect on tubulin dissociation/ association rates, the frequency of transitions to rapid shorten- ing phases (catastrophe), or both. To gain insight into the mech- anism of MT stabilization by mDia2, we tested whether mDia2 affected individual parameters of MT assembly/disassembly by monitoring the behavior of individual MTs assembled from 530 JCB • VOLUME 181 • NUMBER 3 • 2008