crescentinSAT-TC is partially defective both in forming full-length ﬁlamentous structures and in mediating cell curvature. n 212 Cabeen et al. Since crescentinDT was not TC-tagged, we used anti- crescentin immunoﬂuorescence microscopy to visualize this mutant. Unlike wild-type crescentin (Fig. 3b), cres- centinDT failed to produce a consistent ﬁlament-like local- ization. Instead, it displayed multiple patterns ranging from wild-type localization (Fig. 3b, arrow) to spotty, punctate localization patterns (Fig. 3b, arrowheads). This variable localization is consistent with the low median cur- vature of this strain population, as cells with a more severe protein localization defect would display straighter mor- phology and cells with a more wild-type localization would display greater curvature. Our results show that different crescentin mutants dis- play localization defects that differ in severity. The diffuse localization patterns of crescentinDN28-79 and crescentinDR are consistent with no visible assembly whatsoever within cells. The rest of the mutants, however, show some form of crescentin structure (short ﬁlaments, detached ﬁlaments, foci or puncta) in at least some cells. We analyzed these structure-forming crescentin mutants in vitro to determine whether their aberrant localization patterns in cells might be related to structural or assembly defects inherent in the mutant crescentin proteins themselves. Solubility and Ultrastructural Analysis of Wild- Type Crescentin In order to analyze the in vitro properties of structure- forming crescentin mutants, we ﬁrst needed to characterize wild-type crescentin as a comparison. We overproduced and puriﬁed recombinant crescentin from E. coli cells (see Materials and Methods); no protein tag was used for puri- ﬁcation so not to affect the sequence and assembly proper- ties of crescentin. We then ﬁrst determined the assembly properties of wild-type, untagged crescentin using a solu- bility assay. For this assay as well as for the electron mi- croscopy analysis described below, we used a protein concentration of 4 lM. Quantitative immunoblotting yielded estimates of crescentin abundance at 500–1000 Fig. 3. Localization of crescentin domain mutants. (a) Differ- ential interference contrast (DIC) and fluorescence micro- graphs of FlAsH-stained cells producing TC-tagged wild-type or mutant crescentin proteins from a low-copy plasmid in a crescentin-null DcreS background. For crescentinDR (d0), which displayed the weakest staining, a CB15N control (i.e., no TC tag), stained, imaged, and scaled identically to the DR mutant, is shown (e0). The arrow in (g0) shows a curved cell with a bright crescentinSAT-TC structure, and arrowheads indicate cells with short, dimly stained structures. (b) Anti-crescentin immunofluorescence of crescentinDT, which is untagged in order to prevent the generation of an artificial tail. The arrow indicates a cell with a wild-type staining pattern and arrowheads indicate cells with aberrant localization. A wild-type control, stained in parallel, is given at left for comparison. CYTOSKELETON