seem to be hallmarks of bacterial cytoskeletal proteins. In contrast to eukaryotic tubulin and actin, which are extremely conserved even among diverse organisms, bacterial tubulin and actin homologs (e.g., FtsZ or MreB) can vary substantially in sequence across different bacterial species [Erickson, 2007]. Furthermore, there are several distinct families of tubulin homologs (FtsZ, TubZ, BtubA/B) and a surprisingly large number of actin homolog families in the prokaryotic world. In addition to well-studied actin homo- logs such as MreB and ParM, recent work uncovered AlfA, with only 17% identity to MreB [Becker et al., 2006]. AlfA was then used as a starting point to search for more bacte- rial actin homologs too divergent in sequence to be identi- ﬁed using MreB as a reference sequence. This approach uncovered as many as 35 additional families of bacterial actin homologs, all with signature actin motifs [Derman et al., 2009]. All bacterial tubulin and actin homologs examined so far form ﬁlamentous structures in vitro and in vivo [Larsen et al., 2007; Derman et al., 2009], despite being vastly divergent among themselves and from the ca- nonical eukaryotic sequences. It is clear that bacteria have exploited the ﬂexibility of the core tubulin and actin folds to create proteins with properties tailored to speciﬁc func- tions as diverse as cell shape maintenance, plasmid segrega- tion, and cell division [Cabeen and Jacobs-Wagner, 2010]. The discovery of bacterial intermediate ﬁlament (IF)-like proteins [Ausmees et al., 2003; Bagchi et al., 2008] further substantiates the notion that core cytoskeletal motifs are adaptable. Eukaryotic IF proteins are typiﬁed by their pres- ence in metazoa but not plants, protists or fungi. Two meta- zoan IF systems can be distinguished: the nuclear lamins, which form a two-dimensional meshwork underlying the inner nuclear membrane, and cytoplasmic IF proteins, which, in humans, represent a diverse group of more than 70 ﬁbrous proteins with various cellular functions. Both lamins and cytoplasmic IF proteins exhibit a core architec- ture built around a coiled-coil-forming ‘‘rod" domain [Herrmann et al., 2009]. Most notably, while the N-termi- nal ‘‘head" and C-terminal ‘‘tail" domains can differ sub- stantially in length, sequence, and structure, the rod domain always displays an arrangement of distinct coiled- coil motifs of largely conserved length separated by short linkers [Herrmann et al., 2009]. The ﬁbrous nature of IF proteins makes full-length proteins refractory to crystalliza- tion, but their shared coiled-coil domain architecture, and crystallography of protein fragments [Strelkov et al., 2004], indicates that they all fold into a similar three-dimensional coiled-coil structure [Herrmann et al., 2009]. The founding member of the bacterial IF-like family, crescentin, forms a single ﬁlamentous structure along the inner curvature of the crescent-shaped bacterium Caulobacter crescentus [Ausmees et al., 2003; Cabeen et al., 2009]. Without crescentin, C. crescentus cells lose their cur- vature and become straight rods [Ausmees et al., 2003]. Similar to IFs [Yoon et al., 2001], the crescentin structure is n 206 Cabeen et al. very stable in vivo, displaying little to no subunit turnover along its length [Charbon et al., 2009; Esue et al., 2010]. New crescentin molecules are incorporated all along the length of the existing structure [Charbon et al., 2009]. In vitro, His-tagged crescentin forms IF-like ﬁlaments [Ausmees et al., 2003], and the rheological properties of puriﬁed cres- centin solutions resemble those of ﬁlament networks com- posed of the eukaryotic IF vimentin [Esue et al., 2010]. The current model for how the crescentin structure works in vivo proposes that the crescentin structure resists strain (a prop- erty characteristic of IF) produced by cell elongation and thereby mechanically constrains cell wall synthesis to impart cell curvature [Cabeen et al., 2009; Jiang and Sun, 2010]. At the sequence level, crescentin is more similar to eukaryo- tic IF proteins (such as cytokeratin 19, with 25% identity and 40% similarity; [Ausmees et al., 2003]) than MreB or FtsZ is to actin or tubulin [Erickson, 2007], although this is not entirely surprising given the repeated nature and the occur- rence of the coiled-coil motif in both crescentin and IF pro- teins. Crescentin also shares the typical tripartite IF-like domain architecture, with a coiled-coil rod domain inter- rupted by short linker sequences and ﬂanked by short head and tail domains [Ausmees et al., 2003] (Fig. 1a). Even a stut- ter, a small interruption in the characteristic coiled-coil heptad repeat pattern in coil 2 of eukaryotic IF proteins [Herrmann et al., 2009], is present in crescentin [Ausmees et al., 2003] (Fig. 1a). However, crescentin lacks the conserved ‘‘IF-consen- sus motifs" at either end of the rod domain, although a few eu- karyotic IF proteins, such as phakinin and ﬁlensin, diverge substantially from the consensus [Herrmann et al., 2000, 2009]. Importantly, it is unclear whether the IF-like protein organization of crescentin is important for crescentin function. Several mutagenesis studies on eukaryotic IF proteins have examined the roles of speciﬁc regions (e.g., head, linker L1, stutter, and tail) in IF assembly in vitro using puriﬁed proteins. In some cases, in vivo assembly was examined by looking at the ability of mutated proteins to incorporate into (or disrupt) the pre-existing IF network in cell lines, or to form ﬁlaments in IF-free cell lines. However, structure-function relationship studies have gen- erally been limited to known disease-related point muta- tions [Ba¨r et al., 2005, 2006, 2010], most likely because of the lack of good functional assays in cultured cells. Cells lacking cytoplasmic IF proteins do not exhibit any discernable defects in growth, colony-formation, or mor- phology [Venetianer et al., 1983; Hedberg and Chen, 1986]. As for nuclear lamins, only dominant effects of mutant proteins on the endogenous lamins have typically been examined [Spann et al., 1997; Schirmer et al., 2001]. Functional studies are complicated by some IF proteins being essential for viability (e.g., lamins), the ex- istence of redundancies among IF proteins (e.g., keratins) or the complexity of mutant phenotypes at the tissue or organismal level [Harborth et al., 2001; Zhou et al., 2003; Herrmann et al., 2007]. In contrast, the CYTOSKELETON