RESEARCH ARTICLE Cytoskeleton, April 2011 68:205–219 (doi: 10.1002/cm.20505) VC 2011 Wiley-Liss, Inc. The Domain Organization of the Bacterial Intermediate Filament-Like Protein Crescentin is Important for Assembly and Function Matthew T. Cabeen,1† Harald Herrmann,2 and Christine Jacobs-Wagner1,3,4* 1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 2Functional Architecture of the Cell Group, German Cancer Research Center, Heidelberg, Germany 3Section of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 4Howard Hughes Medical Institute, Yale University, New Haven, Connecticut Received 19 January 2011; Revised 1 February 2011; Accepted 2 February 2011 Monitoring Editor: Pekka Lappalainen Crescentin is a bacterial ﬁlament-forming protein that exhibits domain organization features found in meta- zoan intermediate ﬁlament (IF) proteins. Structure-func- tion studies of eukaryotic IFs have been hindered by a lack of simple genetic systems and easily quantiﬁable phenotypes. Here we exploit the characteristic localiza- tion of the crescentin structure along the inner curvature of Caulobacter crescentus cells and the loss of cell curva- ture associated with impaired crescentin function to ana- lyze the importance of the domain organization of crescentin. By combining biochemistry and ultrastruc- tural analysis in vitro with cellular localization and func- tional studies, we show that crescentin requires its distinctive domain organization, and furthermore that different structural elements have distinct structural and functional contributions. The head domain can be func- tionally subdivided into two subdomains; the ﬁrst (amino-terminal) is required for function but not assem- bly, while the second is necessary for structure assembly. The rod domain is similarly required for structure assem- bly, and the linker L1 appears important to prevent run- away assembly into nonfunctional aggregates. The data also suggest that the stutter and the tail domain have Additional Supporting Information may be found in the online version of this article. Abbreviations used: GFP, green ﬂuorescent protein; HEPES, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid; IF, intermediate ﬁlament; MWCO, molecular weight cut-off; LB, lysogeny broth; PAGE, polyacrylamide gel electrophoresis; PIPES, piperazine-N,N0- bis(2-ethanesulfonic acid); SDS, sodium dodecyl sulfate; TEM, transmission electron microscopy. †Matthew T. Cabeen’s present address is Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts. *Address correspondence to: Christine Jacobs-Wagner, KBT 1032, Yale University, PO Box 208103, New Haven, CT 06520-8103, USA. E-mail: christine.jacobs-wagner@yale.edu Published online 28 February 2011 in Wiley Online Library (wileyonlinelibrary.com). critical functional roles in stabilizing crescentin struc- tures against disassembly by monovalent cations in the cytoplasm. This study suggests that the IF-like behavior of crescentin is a consequence of its domain organiza- tion, implying that the IF protein layout is an adaptable cytoskeletal motif, much like the actin and tubulin folds, that is broadly exploited for various functions through- out life from bacteria to humans. VC 2011 Wiley-Liss, Inc. Key Words: crescentin, coiled-coil, cell curvature, Caulo- bacter crescentus, bacterial cytoskeleton Introduction Bnotion acteria that have has a only complex recently subcellular become apparent architecture, thanks a to advances in microscopy techniques. We now know that many important cellular functions are performed by coun- terparts of eukaryotic cytoskeletal proteins [Pogliano, 2008; Graumann, 2009; Cabeen and Jacobs-Wagner, 2010]. For instance, the tubulin homolog FtsZ has an essential role in cell division, whereas actin homologs such as MreB and ParM are important for rod cell shape main- tenance and plasmid segregation. The characterization of bacterial tubulin and actin homologs as cytoskeletal pro- teins has historically followed a common path. Low pri- mary sequence similarity between bacterial and eukaryotic cytoskeletal proteins initially hampered their identiﬁcation. Instead, the presence of small, structurally signiﬁcant motifs important for nucleotide binding yielded the ﬁrst hints that these proteins were cytoskeletal [Bork et al., 1992; de Boer et al., 1992; RayChaudhuri and Park, 1992; Mukherjee et al., 1993]. Subsequent structural anal- ysis showed that these proteins indeed shared a common fold with eukaryotic tubulin and actin [Lo¨we and Amos, 1998; van den Ent et al., 2001]. Continued study of bac- terial tubulin and actin homologs revealed themes that 205 n