Fig. 5. Solubility of crescentin mutants. Comparison of wild- type and mutant solubility at pH 6.5, 7.5, and 8.5 under the noted conditions. Bars show the proportion of crescentin pelleted after centrifugation for 15 min at 135,000 3 g. The average of three experiments is shown, and error bars repre- sent SEM. on solubility except at the highest concentration tested (200 mM), where they increased solubility slightly (Fig. 4a). Our pelleting assays measured insolubility rather than giving data about the size or shape of the insoluble cres- centin material. To examine the structures formed, we used transmission electron microscopy (TEM) of nega- tively stained crescentin samples. Crescentin formed IF- like ﬁlaments of 8–10 nm width at pH 6.5 (Fig. 4b, arrowheads) as well as 17–20 nm-wide ﬁlament pairs (Fig. 4b, arrows) or larger bundles ( 50 nm, Fig. 4b, asterisk), indicating a propensity for lateral association. Filaments were also readily visible in ﬁve-fold diluted crescentin n 214 Cabeen et al. samples (0.8 lM; data not shown). Addition of 5 mM MgCl2 at pH 6.5 increased the appearance of 20 nm- wide pairs (Fig. 4c, arrows) and larger bundles (Fig. 4c, asterisks), consistent with the divalent Mg2þ ions having a bundling effect. Wider ﬁlamentous structures in the pres- ence of Mg2þ were also predominant at pH 7.5 (Fig. 4d), while at pH 8.5, ﬁnely reticulated ﬁlament networks were most commonly observed (Fig. 4e). Crescentin ﬁlaments were also bent along their length, thereby resembling the characteristic ﬂexibility of eukaryotic IF proteins [Herr- mann et al., 2007]. Solubility and Ultrastructural Analysis of Crescentin Mutants We next puriﬁed each (tag-less) crescentin mutant that formed some sort of structures visible by ﬂuorescence mi- croscopy in vivo (Fig. 3) and analyzed their solubility under a subset of the conditions used for wild-type cres- centin. We also included crescentinDN27 in this study, which forms an intracellular ﬁlamentous structure [Cabeen et al., 2009, 2010] but has not yet been studied in vitro. We varied pH over the same range of buffer concentra- tions and tested the effects of Kþ and Mg2þ ions at their highest concentrations (200 mM and 5 mM, respectively), where they exerted the clearest effect on wild-type cres- centin solubility. As with wild-type crescentin, lower pH strongly favored pelleting of all the crescentin mutants, with signiﬁcant as- sembly at pH 7.5 and 8.5 occurring only in the presence of 5 mM Mg2þ ions (Fig. 5). Strikingly, crescentinDL1 was more insoluble than wild-type crescentin or any of the other mutants in many conditions. It was nearly com- pletely insoluble across all conditions at pH 6.5, even in weak buffer (5 mM) in the absence of monovalent or divalent cations (Fig. 5). CrescentinDL1 also showed slightly enhanced insolubility at pH 7.5 relative to wild- type and the other mutants. The high insolubility of cres- centinDL1 implies strong assembly or aggregation that is consistent with the compact, focus-like localization (Fig. 3a-f0 ) and severe dominant-negative effect of crescentinDL1 in cells (Fig. 2). Surprisingly, TEM of crescentinDL1 revealed ﬁlamentous structures rather than amorphous aggregates. In weak 5 mM buffer at pH 6.5, ﬁlaments of varying width were observed, with single 9 nm ﬁlaments (Fig. 6a, arrows) as well as ﬁlaments with twice or three times that width (Fig. 6a, arrowhead and asterisk, respectively). Filaments were often much shorter than those formed by wild-type crescentin (Fig. 6a, asterisk; compare to Fig. 5b). In 50 mM buffer at pH 6.5, tapered structures with varying width were common, with the narrowest ﬁlaments about 10–12 nm wide (Fig. 6b, arrowhead). In the presence of 200 mM KCl, crescentinDL1 formed large, rough, aggrega- tive structures that appeared to contain globular particles CYTOSKELETON