Figure 7. FH1FH2mDia2 stabilizes MTs by decreasing rates of MT disassembly. (A–D) Disassembly assays of MTs elongating from axonemal fragments and induced to disassemble by dilution in the presence or absence of the indicated concentrations of His-FH1FH2mDia2 or buffer alone. The rate of MT shorten- ing was monitored by real-time video DIC microscopy. (A) A series of micrographs taken from a real-time video recording of MTs undergoing shortening. Arrowheads point to MT ends shortening in the presence of 2 μM His-FH1FH2mDia2 (mDia2) or buffer alone. Time is shown in seconds. (B) Histogram of the frequency of ﬁnal shortening velocities of MT plus and minus ends after dilution into 2 μM His-FH1FH2Dia2 or buffer alone. Arrows indicate means for each condition. Data are normalized percentages of total MTs (to account for differences in sample size; n ≥ 53 ends for each condition). Velocities >90 μm/min are pooled in the last bin. (C) Graphs showing the dependence of shortening velocity of MT plus and minus ends on concentration of His- FH1FH2mDia2 (mDia2). Data are mean ± SE of shortening velocities (n ranges from 3 to 27 for each data point). (D) Plots of life histories of representative MT plus ends induced to disassemble by dilution into buffer alone or into buffer with 2 μM His-FH1FH2mDia2. Bar, 5 μm. MT stability by mDia2 was also observed in assays with perfu- sion chambers where the concentration of free tubulin is essen- tially negligible after dilution, we suspected that stabilization would be mediated by inhibition of polymer dynamics rather than by stimulation of dimer assembly. Consistent with this idea, we found no evidence for mDia2 interacting with tubulin dimers and observed a small, but significant, decrease in the rate of MT polymerization when mDia2 was incubated with tubulin during assembly (Table I). An even larger effect of mDia2 was observed on rates of MT shrinkage and, in a small percentage of cases, this effect led to MTs that exhibited shrinkage rates near zero. The ability of mDia2 to strongly slow MT shrinkage rates is presumably related to mDia’s role in generating stable MTs in vivo that do not shrink for extended intervals and behave as if they are capped (Cook et al., 1998; Infante et al., 2000; Palazzo et al., 2001). In this respect, mDia2’s effect on MTs is distinct from traditional MAPs, which increase MT assembly rates without significantly interfering with MT shrinkage rates under similar experimental conditions (Drechsel et al., 1992; Pryer et al., 1992; Vasquez et al., 1994). Although mDia2’s ability to stabilize MTs in vitro is con- sistent with its activity in generating stable MTs in vivo, the in vitro experiments with axonemes showed that mDia2 did not completely block MTs from growing or shrinking, respectively, as is observed for Rho/mDia-stabilized MTs in vivo. One possi- bility is that full-length mDia2 has significantly greater activity toward MTs than the FH1FH2 fragment we used in the in vitro experiments. It also seems likely that the capping of MTs ob- served in vivo requires additional cellular factors that function with mDia. Indeed, APC and EB1, two MT binding proteins themselves, interact with mDia and have been implicated in generating stable MTs induced by LPA (Wen et al., 2004). Also, mDia regulation of GSK3 through novel PKCs has been recently shown to promote MT stabilization in migrating fibro- blasts (Eng et al., 2006). It is possible that these factors, and maybe others yet to be discovered, contribute to cap MTs 532 JCB • VOLUME 181 • NUMBER 3 • 2008