plasmid under the native promoter in a crescentin-null background as the only source of crescentin in the cell. Production of each mutant was conﬁrmed by anti-cres- centin Western blotting, with the less intense staining of the rod mutant (crescentinDR) likely due to epitope loss from the large deletion (Fig. 1b). We used automated cell curvature analysis to assess the function of each mutant (Fig. 1c). The head domain of eukaryotic IF proteins, despite high variability in length and amino acid sequence, is gen- erally regarded as essential for IF function. The head is required for polymerization of both nuclear [Heitlinger et al., 1992] and cytoplasmic IF beyond oligomeric subu- nits in vitro [Hatzfeld and Burba, 1994; Herrmann et al., 1996] and for ﬁlament formation in vivo [Raats et al., 1990; Beuttenmu¨ller et al., 1994; Spann et al., 1997]. Furthermore, phosphorylation or point mutations in the head adversely affect self-assembly [Geisler and Weber, 1988; Sharma et al., 2009]. We therefore analyzed the function of the head domain of crescentin in C. crescentus curvature. Importantly, we distinguished between two sub- domains in the crescentin head. We had previously shown that the amino-terminal N27 subdomain (Fig. 1a) is not necessary for crescentin structure formation in vivo but rather is required for the association of the crescentin structure with the cell wall and hence cell curvature [Cabeen et al., 2009]. Here, we generated an in-frame de- letion that maintains the N27 region but removes the re- mainder of the head (Fig. 1a), and analyzed the ability of the resulting mutant protein (termed crescentinDN28-79) to mediate cell curvature. Cells producing crescentinDN28-79- TC displayed no detectable curvature (Fig. 1c), indicating that this deletion completely abrogated crescentin function. In vimentin, a functional deletion of linker L1 yields a protein able to assemble beyond small oligomers into amorphous aggregates but unable to elongate into ﬁla- ments in vitro [Herrmann et al., 1999]. The short, 8-resi- due length of linker L1 in vimentin was effectively transformed into part of the rod by the addition of three residues that restore a coiled-coil sequence [Herrmann et al., 1999]. Given the longer length of crescentin L1 (21 residues), we utilized a deletion mutant (D122-142) lack- ing L1 and fusing coils 1A and 1B to each other. We had previously reported that this crescentinDL1 mutant was nonfunctional and in fact had substantial dominant-nega- tive activity [Cabeen et al., 2009]; however, we had not quantitatively analyzed the curvature of a strain producing crescentinDL1-TC. This analysis conﬁrmed that crescent- inDL1-TC bears no measurable cell curvature function (Fig. 1c). The rod domain, with its coiled-coil-forming regions, would be predicted to have a key role in assembly. Few studies have analyzed the role of the rod domain, but nu- clear lamin B1 deleted for all but the ﬁrst and last ﬁve n 210 Cabeen et al. coiled-coil heptad repeats in the rod domain resulted in a dominant-negative effect, causing a nuclear morphology defect in cultured cells [Schirmer et al., 2001]. As lamin B1 is essential for viability [Harborth et al., 2001], the functional properties of rod-deleted lamin B1 alone have not been examined. We generated an analogous deletion of all but the ﬁrst and last 5 coiled-coil heptads in the rod domain of crescentin. Consistent with an important func- tional role, crescentinDR-TC induced no detectable cell curvature (Fig. 1c). Removal of the coil 2B stutter in vimentin produced a protein capable of the initial stages of assembly but defec- tive in ﬁlament elongation in vitro, suggesting that the stutter plays an important role in ﬁlament assembly [Herr- mann et al., 1999]. In vimentin, stutter removal was accomplished by the insertion of three amino acids (ser- ine, alanine, and threonine; S-A-T) that restored the coiled-coil heptad repeat pattern [Herrmann et al., 1999]. We employed the same strategy, as a S-A-T insertion in crescentin restores the coiled-coil heptad repeat pattern and eliminates the stutter, according to the MARCOIL prediction algorithm [Delorenzi and Speed, 2002]. Cells producing the resulting crescentinSAT-TC showed no measurable curvature (Fig. 1c), suggesting that the stutter in crescentin is functionally important as well. Finally, we created a C-terminal truncation of crescentin that lacked the tail region. The predicted tail region of crescentin is very short and a-helical. In vimentin, dele- tion of the tail resulted in greater variation in ﬁlament width in vitro, and thicker ﬁlaments in cells, compared to the wild-type [Herrmann et al., 1996]. However, tail- deleted vimentin could still form ﬁlament networks in transfected cells [Rogers et al., 1995], consistent with a minor role for the tail in assembly. CrescentinDT lacked 13 C-terminal amino acids, and we chose not to TC-tag crescentinDT in order to avoid generating an artiﬁcial C- terminal tail. CrescentinDT, unlike the other crescentin mutants, was able to support some curvature. However, cells producing crescentinDT displayed on average only a fraction of the curvature (26%) of an isogenic strain pro- ducing wild-type crescentin-TC (Fig. 1c), suggesting a substantial defect in cell curvature function. The fact that crescentinDT was the only mutant to retain some function is consistent with the tail playing a less crucial role, as observed with eukaryotic IF proteins. On the other hand, the head, linker L1, rod, and stutter are essential for cres- centin function in vivo, consistent with eukaryotic IF studies. Domain Mutants Interfere with the Function of Wild-Type Crescentin Production of rod-deleted lamin B1 in cultured cells, or microinjection of head-deleted lamin A into Xenopus oocytes, negatively affects the localization and function of CYTOSKELETON