Fig. 2. Cell curvature effect of crescentin mutants in the presence of wild-type crescentin. Plasmid-encoded crescentin or crescentin mutants were produced in a background (CB15N) producing wild-type crescentin from the chromosome and were assessed for their effect on cell curvature. The curvature values are relative to wild-type CB15N cells (lacking plasmid). Error bars represent SEM of three experiments; each experiment examined, on average, over 1200 cells. wild-type lamins [Spann et al., 1997; Schirmer et al., 2001]. Such dominant-negative activity is likely driven by formation of disruptive heterocomplexes between the mu- tant and wild-type proteins and thus informs about the interaction strength between wild-type and mutant pro- teins. Do crescentin mutants have a similar negative effect on wild-type crescentin? To address this question, we introduced plasmid-encoded crescentin-TC mutants into the wild-type CB15N background producing wild-type crescentin from the chromosome. For comparison, pro- duction of wild-type crescentin-TC from the same low- copy-number plasmid resulted in a small but quantiﬁable hypercurvature relative to the wild-type CB15N lacking plasmid (Fig. 2), consistent with overproduction leading to cell hypercurvature [Cabeen et al., 2009]. In contrast, production of four of the ﬁve crescentin mutants resulted in decreased curvature values (Fig. 2), showing that they possess dominant-negative activity. CrescentinDL1-TC, which has previously been reported as a potent dominant negative [Cabeen et al., 2009], had the strongest effect with a near-complete abrogation of curvature. Crescent- inDN28-79-TC and crescentinSAT-TC both had an interme- diate effect, reducing curvature values to roughly half of wild-type, while crescentinDR-TC had the mildest negative effect, slightly reducing curvature from the wild-type value. These results show that despite their lack of func- tion when expressed on their own, these crescentin mutants are not inert. Rather, they appear to retain the ability to interact with wild-type crescentin to affect its function. The milder effect of crescentinDR-TC is consist- ent with the idea that disruption occurs through hetero- CYTOSKELETON complex formation, as interactions between wild-type crescentin and crescentinDR-TC are expected to be weak- ened by the much shorter coiled-coil region of the rod- deleted mutant. Conversely, the longer uninterrupted coiled-coil of crescentinDL1-TC might strengthen hetero- complexes, leading to its strong disruptive effect. Interestingly, crescentinDT, which on its own could sup- port only a small fraction of wild-type curvature function (Fig. 1c), was able to induce cell hypercurvature at nearly the level of wild-type crescentin when produced in combi- nation with endogenous crescentin (Fig. 2). This result suggests that crescentinDT, while deﬁcient in establishing a functional structure on its own, can ‘‘strengthen" a wild- type crescentin structure and enhance cell curvature. In this case, heterocomplexes between crescentinDT and wild- type crescentin may be functional rather than disruptive. Localization of Crescentin Mutants The crescentin mutants all displayed defects in cell curva- ture (Fig. 1c). Could localization defects be responsible for these impairments? Wild-type crescentin-TC stained with FlAsH displayed the expected ﬁlament-like localiza- tion along the inner curvature of C. crescentus cells (Figs. 3a-a0). Each of the crescentin mutants, meanwhile, had localization defects ranging from mild to severe. Crescent- inDN27-TC formed curved, detached ﬁlamentous structures in some cells [Cabeen et al., 2009], while many cells dis- played a diffuse localization (Fig. 3a-b0). Both crescent- inDN28-79-TC and crescentinDR-TC gave diffuse cytoplasmic signals (Fig. 3a-c0 and 3a-d0, respectively), suggesting a defect in ﬁlamentous structure assembly. The diffuse ﬂuorescence of crescentinDR-TC, the weakest among the mutants, was more intense than background ﬂuorescence of FlAsH-stained wild-type CB15N cells pro- ducing no TC-tagged protein (Fig. 3a-e0), arguing that it represents a true localization pattern for crescentinDN28-79- TC and crescentinDR-TC. CrescentinDL1-TC frequently displayed ﬂuorescent foci, as previously reported [Cabeen et al., 2009]; shown in Fig. 3a-f0 for comparison), in agreement with an aggregative phenotype wherein cres- centinDL1-TC molecules form clusters. CrescentinSAT-TC was characterized by three distinct localization patterns. About half the cells (49.5%, n ¼ 384) showed only diffuse ﬂuorescence, while most other cells (47.9%) contained short, dimly staining crescentin structures that had no evident ability to induce cell curva- ture (Fig. 3a-g0, arrowheads). The remaining small fraction of cells (2.6%) displayed curvature that was associated with short, brightly staining crescentinSAT-TC structures at the site of curvature (Fig. 3-g0, arrow). The curvature of these cells often appeared highly localized, with a sharp curve at the location of the crescentinSAT-TC structure. The low frequency of curved cells and the short appear- ance of the crescentinSAT-TC structures suggest that Analysis of Crescentin Domain Organization 211 n