with 20–30 nm diameter (Fig. 6c, arrowheads). Such structures would be consistent with the insolubility of crescentinDL1 in 200 mM Kþ (Fig. 5) but are clearly dis- similar to normal ﬁlamentous networks. With 5 mM Mg2þ ions, broad, tapered structures formed (Fig. 6d), consistent with enhanced lateral association mediated by the divalent cations. With Mg2þ at pH 7.5, similar broad structures predominated (Fig. 6e). Among the other mutants, crescentinDN27, crescentin- SAT, and crescentinDT were notable for their high sensitiv- ity to 200 mM Kþ ions, which substantially reduced their solubility at pH 6.5 (Fig. 5). CrescentinDN27 was also extremely soluble in weak (5 mM) buffer at pH 6.5, and was the most soluble mutant, along with crescentinSAT, in Mg2þ at pH 7.5 (Fig. 5). The heightened sensitivity of crescentinDN27, crescentinSAT, and crescentinDT to potas- sium may help explain the relatively high frequency of cells with diffuse crescentin localization in these three mutants (Fig. 3). Cytoplasmic Kþ ions could lower the favorability of assembly to the point where only a fraction of cells successfully assemble a crescentin structure (see Discussion). Consistent with the ﬁrst 27 amino acids of crescentin primarily functioning in membrane attachment of cres- centin structures rather than in ﬁlament formation in vivo [Cabeen et al., 2009], puriﬁed crescentinDN27 assembled into ﬁlaments largely indistinguishable from wild-type. At pH 6.5, single crescentinDN27 ﬁlaments of 8–9 nm width were commonly observed (Fig. 6f, arrows), as well as bun- dles with double or triple this width (Fig. 6f, arrowhead and asterisk, respectively) and larger loose bundles. Similar to wild-type crescentin, addition of 5 mM Mg2þ ions at pH 6.5 resulted in smooth but thicker crescentinDN27 ﬁla- ments of about 17 nm (Fig. 6g, arrowhead), though single 9-nm ﬁlaments were still visible (Fig. 6g, inset, arrow). In the presence of 200 mM Kþ ions, crescentinDN27 formed rough, branched structures with a 22-nm width (Fig. 6h, arrow). The ﬁlaments observed for crescentinDN27 are in accord with earlier results from our laboratory using His- tagged crescentinDN27 assembled at pH 7.0 [Ausmees et al., 2003]. The previous analysis used crescentinDN27, instead of wild-type crescentin as we originally assumed, because of a mis-annotated start site in the genome sequence database; we have since conﬁrmed the correct start site using mass spectrometry (Supporting Informa- tion Fig. S1) and have updated the public database. In any case, loss of the N27 region of crescentin does not substantially alter ﬁlament assembly properties in vitro. At pH 6.5, crescentinSAT formed structures that were characterized by axial striations displaying a 57.1 6 0.7 nm repeat (n ¼ 25; Fig. 6i, inset) most often in ﬁlament bundles. Individual ﬁlaments with 9 nm width were also visible (Fig. 6i, inset, arrowhead), as were bundles 40 nm in width (Fig. 6i, arrows). No striations were seen in the smooth crescentinSAT ﬁlament bundles polymerized in n 216 Cabeen et al. the presence of magnesium ions (Fig. 6j). However, at pH 7.5 in the presence of Mg2þ, spike-like paracrystals were the predominant species, with an axial repeat of 58.7 6 0.3 nm (n ¼ 30; Fig. 6k, inset). The thickness of the structures suggests that a possible function of the stutter is to prevent excessive bundling of crescentin ﬁlaments. Such paracrystalline structures have been observed in the pres- ence of divalent cations for truncation mutants of a eu- karyotic IF protein, glial ﬁbrillary acidic protein [Quinlan et al., 1989; Stewart et al., 1989] and for tailless Xenopus vimentin [Eckelt et al., 1992]. CrescentinDT formed long, smooth ﬁlaments at pH 6.5 that were similar in appearance to wild-type crescentin ﬁl- aments, except that the narrowest structures observed were 17–20 nm wide (Fig. 6l, arrowheads) rather than 8–10 nm. Addition of Mg2þ ions at pH 6.5 caused ﬁlament bundling and the appearance of striations with 55.6 6 0.6 nm spacing (n ¼ 30; Fig. 6m). The crescentinDT ﬁla- ment bundles often appeared tapered at their ends. With magnesium at pH 7.5, no striations were visible in the ﬁl- amentous structures with varying width (Fig. 6n). The similarity of crescentinDT to wild-type in vitro is consist- ent with it being the most functional of all the mutants. Discussion In this study, we used the simple and tractable organism C. crescentus with two goals in mind. First, we aimed to discover whether the IF-like domains of crescentin are in fact important for its structure and function. Second, we analyzed different domain mutants to learn whether spe- ciﬁc domains have distinct structural or functional roles. For this, we utilized the advantages of our bacterial model system—genetic tractability, a quantiﬁable cell curvature phenotype and a well-deﬁned cellular localization pattern. Deletion or abrogation of either head subdomain, linker 1, rod, stutter, or tail all had profound effects on cres- centin function in vivo, indicating that many elements of the IF-like organization of crescentin are indeed function- ally important. Moreover, each mutation had a distinct effect, in accord with different domains having different roles. Based on our data, we suggest the following functions for each element. Mutants lacking the N28-79 head sub- domain and rod domains display no structure in vivo, suggesting a fundamental role for these elements in ﬁla- mentous structure assembly. Linker L1 may play the op- posite role, preventing the runaway polymerization of crescentin that disrupts its normal ﬁlamentous cellular localization and function. It may fulﬁll this function sim- ply by providing an interruption in the coiled-coil rod do- main. Linker L1 also appears to be like the stutter in that both these regions lower the propensity for lateral associa- tion, most apparent in vitro by the broad structures formed by crescentinDL1 and paracrystal formation by CYTOSKELETON