crescentinSAT. It is clear that crescentinDN27, crescentinSAT, and crescentinDT are capable of assembling into long ﬁla- ments in vitro. Therefore, the observations that crescentin- SAT and crescentinDT typically form only short structures in cells, and that crescentinDN27 and crescentinSAT often show diffuse localization, are not best explained by a structural defect. Instead, our in vitro data suggest that the tail, stutter, and N27 regions stabilize crescentin against disassembly in the presence of Kþ ions. This is not a trivial role, as several studies have estimated E. coli cytoplasmic potassium concentration at about 200 mM [Prigent-Combaret et al., 1999; Outten and O’Halloran, 2001; Shabala et al., 2009]. Assuming a similar concentra- tion in C. crescentus, this level would be adequate for a substantial inhibition of in vivo crescentin assembly in these three mutants. Cytoplasmic Kþ ions may favor dis- assembly of these mutants so that full elongation of cres- centin structures is very rare, perhaps explaining the high frequency of diffuse localization or short structures in cells producing these mutants. Collectively, our study suggests that in order to function, crescentin must meet multiple criteria. First, the protein must assemble into a structure, a function that appears to require the N28-79 and rod domains. Next, it must be able to assemble even in the presence of Kþ ions, a function to which the N27, stutter, and tail regions contribute. How- ever, crescentin must not polymerize excessively or bundle too much—the L1 linker and the stutter assist this function. Finally, crescentin must be able to associate with the mem- brane, a function served by the N27 region. IF proteins are found in all animal cells, and the Human Intermediate Filament Database (www.interﬁl.org/diseases) lists 90 human diseases that have been linked to mutations in IF genes. Still, IFs are the least understood of the three major eukaryotic cytoskeletal systems. Research efforts have been hampered by the large number of IF proteins in mammalian systems, the possible functional redundancy between speciﬁc IFs, and the lack of powerful genetic model systems in which to study them. Our study shows that crescentin not only bears but requires its characteristic IF features—head, rod, linker 1, stutter, and tail—for structure and function. This structural similarity to IF proteins, combined with the IF-like dynamic and mechanical properties of crescentin in vitro and in vivo [Cabeen et al., 2009; Charbon et al., 2009; Esue et al., 2010], establishes crescentin as a valuable bacterial model for the study of a broad family of IF proteins that we propose includes both eukaryotic and prokaryotic members. By the term ‘‘family,’’ we mean a group of proteins that shares a characteristic set of IF-like biochemical, mechani- cal, and cytoskeletal properties deriving from a particular protein organization. We do not imply any particular evo- lutionary relationship between crescentin and animal IF proteins, as we lack sufﬁcient data to make deﬁnitive statements. While it seems clear that both crescentin and animal IF proteins derive from a bacterial coiled-coil-rich CYTOSKELETON protein ancestor, how IF-like domain organization arose in each case remains obscure. It is formally possible that it arose in bacteria, further evolved into a more constrained structural organization in eukaryotes, and was then lost in many species. Alternatively, IF-like organization may have independently evolved in bacterial and animal cells through convergent evolution of a basic coiled-coil motif. Finally, we cannot rule out lateral gene transfer from ani- mal cells to bacteria, although the current aquatic, nutri- ent-poor ecological niche of C. crescentus does not suggest close interactions with animal cells. Importantly, this IF family is a subset of a coiled coil- rich protein superfamily, and eukaryotic IF proteins are a further, more constrained, subset. In this way, the IF fam- ily resembles the actin family, which is a subset of a nucle- otide-binding protein superfamily, and includes both the divergent bacterial actin proteins and the highly conserved eukaryotic actin [Bork et al., 1992; Derman et al., 2009]. There are some variations between crescentin and eukary- otic IF proteins, in coiled-coil motif length and in amino acid sequences at either end of the rod domain, and these differences parallel the distinction between bacterial and eukaryotic tubulin and actin homologs. Just as bacterial actins and tubulin homologs display a core set of motifs in order to function, crescentin contains a core set of IF protein features that allow its function. We speculate that during evolution, the actin, tubulin, and IF motifs have been selected for their utility and versatility as cytoskeletal elements. It will surely be interesting to explore how bac- teria use other variations of the IF architecture [Izard, 2006; Bagchi et al., 2008] to fulﬁll different cellular roles. Acknowledgments We thank Godefroid Charbon for the kind gift of immu- noprecipitated crescentin-FLAG for the mass spectrometry analysis, and Mark Mooseker, Thomas Pollard, and An- thony Koleske for technical advice. FlAsH reagent was a gift of Doug Weibel (Univ. Wisconsin-Madison). Thanks are also due to members of the Jacobs-Wagner laboratory for helpful discussions and for critical reading of this manuscript. M.T.C was supported in part by an EMBO Short-Term Fellowship, the NSF GRFP and the Mustard Seed Foundation. H.H. acknowledges support from the German Research Foundation (DFG HE 1853/5-4). This work was supported by NIH grant GM076698 and the Pew Scholars Program in the Biological Sciences spon- sored by the Pew Charitable Trust (C.J.-W.). C.J.-W. is a Howard Hughes Medical Institute Investigator. References Ausmees N, Kuhn JR, Jacobs-Wagner C. 2003. The bacterial cyto- skeleton: an intermediate ﬁlament-like function in cell shape. Cell 115 (6): 705–713. Bagchi S, Tomenius H, Belova LM, Ausmees N. 2008. Intermediate ﬁlament-like proteins in bacteria and a cytoskeletal function in Streptomyces. Mol Microbiol 70 (4): 1037–1050. Analysis of Crescentin Domain Organization 217 n