et al., 2003] using anti-crescentin antibodies at 1:200. Image capture and analysis were performed with Meta- Morph software (Molecular Devices). Cell curvature anal- ysis was performed on phase-contrast images as described [Cabeen et al., 2009, 2010]. Three separate experiments were performed for each strain, and the number of cells analyzed for each experiment ranged from 633 to 2150. Statistical calculations were performed with MATLAB and Microsoft Excel. Immunoblotting Cell extracts (load volumes normalized by culture optical density at 660 nm; OD660) were separated on sodium do- decyl sulfate (SDS)-10% polyacrylamide gels, transferred to Immobilon P membranes (Millipore) using a Bio-Rad Trans-Blot apparatus, and blotted with afﬁnity-puriﬁed anti-crescentin antibodies at 1:10,000. Quantitative im- munoblotting was performed by comparing a standard dilution series of puriﬁed crescentin as standards to wild- type (CB15N) cell extracts from known quantities of cells. Chemiluminescently stained blots were analyzed on a Ty- phoon scanner (GE) and quantiﬁed using ImageQuant. Protein Puriﬁcation Untagged wild-type and mutant versions of crescentin were puriﬁed under identical conditions from E. coli BL21 (DE3) strains carrying the creS (or mutant) coding sequence in pET29a. Cells (0.5–1 L) were grown at 37 C in LB medium to OD600 0.6, then induced with 1 mM isopropyl b-D-1-thiogalactopyranoside for 3 h. Cells were harvested by centrifugation at 3000 g for 15 min at 4 C and frozen at 80 C overnight. Thawed cells were resuspended and lysed in Buffer A (25 mM sodium ace- tate pH 4.5, 8 M urea) and the lysate was cleared at 12,000 g for 30 min. Urea was always freshly prepared in the presence of a mixed-bed ion-exchange resin. Wild- type and mutant versions of crescentin were puriﬁed from the lysate on CM-sepharose (GE) equilibrated with Buffer A, washed with Buffer A, and eluted with a salt gradient (0–300 mM or 0–150 mM NaCl in Buffer A). Cres- centin-containing fractions [determined by SDS-polyacryl- amide gel electrophoresis (PAGE)] were pooled and the urea was removed by 15 min-stepwise dialysis (25 kDa MWCO), halving the urea concentration each time, then overnight dialysis against 2 L pure 5 mM HEPES pH 7.5 or Tris-HCl pH 8.5 (dialysis at pH 6.5 caused precipita- tion). Crescentin and crescentin mutant concentration was determined using a Bradford assay (Bio-Rad). Protein preparations were stored in buffer at 4 C and checked for protein degradation by PAGE before use. Pelleting Assays Crescentin samples (20 lL) at 8 lM were mixed 1:1 with a 2 ‘‘assembly buffer" composed of buffer (PIPES CYTOSKELETON pH 6.5, HEPES pH 7.5, or Tris-HCl pH 8.5) and salt (KCl or MgCl2) as noted; samples at pH 6.5 were kept at 5.4 lM to preserve solubility and mixed 3:1 with a 4 assembly buffer. The 40 lL ﬁnal samples at 4 lM were then allowed to polymerize for 10–15 min at room tem- perature before pelleting at 135,500 g for 15 min at 25 C (Beckman Optima Max-XP, TLA-100 rotor). Super- natant samples (16 lL) were taken from the top of the tube, remaining supernatant was removed, and pelleted material was solubilized in 40 lL 5 mM Tris-HCl pH 8.5, 8 M urea for 10 min. A 16 lL sample of the solu- bilized pellet was then taken. The supernatant and pellet fractions were mixed 4:1 with 5 SDS-PAGE loading buffer, run on 10% polyacrylamide gels, stained with Coomassie Brilliant Blue, and digitally scanned using a ﬂatbed scanner. Relative protein amounts in the superna- tant and pellet fractions were determined by densitometry of inverted images using ImageJ. All experiments were performed in triplicate. Loading known amounts of cres- centin conﬁrmed the accuracy of the densitometry method. Electron Microscopy Samples of crescentin preparations were diluted to 4 lM with 2 or 4 assembly buffer as described above, allowed to polymerize for 10–15 min at room tempera- ture, and applied to freshly glow-discharged 400 mesh carbon-coated copper grids (Electron Microscopy Scien- ces). After allowing proteins to settle for 2–3 min, grids were washed with water three times (water was removed after the last wash by touching the grid to ﬁlter paper) and stained for about 1 min with 1% uranyl acetate. The uranyl acetate was removed using ﬁlter paper and the grids were dried for several hours. The samples were viewed with a JEOL JEM-1230 transmission electron microscope at 80 kV and images were acquired with a Hamamatsu ORCA-HR digital camera. Results Domain Mutants are Impaired in Cell Curvature Function Previous mutagenesis studies on eukaryotic IF proteins have shed light on the role of some elements of the con- served IF domain organization in ﬁlament assembly (as described below). We combined this knowledge to exam- ine in a single study whether several analogous mutations in crescentin affect its function in vivo. Each mutant, with the exception of the tail mutant (crescentinDT; see below), was tagged with a TC tag for visualization inside cells using the ﬂuorescent dye FlAsH [Grifﬁn et al., 1998] since TC tagging, unlike GFP tagging, does not disrupt crescentin function [Ausmees et al., 2003; Cabeen et al., 2009]. All mutants were produced from a low-copy Analysis of Crescentin Domain Organization 209 n