Fig. 1. Functional assessment of crescentin mutants in cell curvature. (a) Schematic of crescentin domain organization. Coiled coil-forming regions are indicated by thick green bars and labeled by amino acid positions at junctions with other features; linkers L1, L12, and L2; N27 and N28-79 subdomains of the head, tail, and stutter are labeled. (b) Western blot of crescentin mutants using a-crescentin. The relatively weak signal of crescentinDR is likely due to epitope loss. Loading was normalized by OD660. (c) The ability of the deletion mutants to curve C. crescentus DcreS cells is shown. Curvature values relative to DcreS cells producing wild-type crescentin-TC from the same low-copy plasmid pMR20 (100%). Error bars represent the standard error of the mean (SEM) of three experiments; each experiment examined, on average, over 1200 cells. characteristic localization of crescentin along the inner curvature of C. crescentus cells and the loss of cell curva- ture associated with a loss of crescentin function provide quantiﬁable assays for in vivo functional studies [Cabeen et al., 2009]. We use this bacterial model system to inves- tigate two questions. Are the IF-like organizational fea- tures of crescentin functionally important, as opposed to merely incidental? Do different features have distinct roles in creating a functional crescentin structure? Materials and Methods Bacterial Culture, Strains, Plasmids, and Mutagenesis C. crescentus was grown in peptone-yeast extract (PYE) or M2G supplemented with 1% PYE (M2Gþ) at 30 C [Ely, 1991]. Plasmids were transformed into C. crescentus by CYTOSKELETON electroporation or conjugation with Escherichia coli strain S17-1 [Ely, 1991]. Strains and plasmids are listed in Table I, and their modes of construction and primer sequences are given in the Supporting Information. Exponentially growing cultures were used for all experiments. Light Microscopy and Cell Curvature Analysis Cells were imaged at room temperature ( 22 C) on a Nikon E1000 microscope ﬁtted with 100 differential in- terference contrast or phase-contrast objectives and a Hamamatsu Orca-ER LCD camera, or a Nikon E80i microscope ﬁtted with similar objectives and a Hama- matsu Orca-IIER LCD camera. Cells were immobilized on 1% agarose-PYE or M2Gþ pads. FlAsH staining of tetracysteine (TC)-tagged crescentin was performed as pre- viously described [Cabeen et al., 2009]. Immunoﬂuores- cence microscopy was performed as described [Ausmees Analysis of Crescentin Domain Organization 207 n