Fig. 4. Wild-type crescentin solubility and ultrastructure. (a) Solubility of purified wild-type crescentin over a range of pH val- ues, buffer concentrations, and KCl and MgCl2 concentrations. Bars show the proportion of crescentin pelleted after centrifugation for 15 min at 135,000 g. The average of three experiments is shown, and error bars represent SEM. (b–e) Transmission elec- tron micrographs of negatively stained wild-type crescentin. Samples (0.2 mg/mL, 4 lM) were in 50 mM PIPES (pH 6.5), HEPES (pH 7.5), or Tris-HCl (pH 8.5). Mg2þ denotes 5 mM MgCl2. (b) Single filaments of 8–10 nm width (arrowheads), pre- sumed pairs with widths of 17–20 nm (arrows), and a 50 nm-wide bundle (asterisk) are denoted. (c) Crescentin sample in the presence of 5 mM Mg2þ. A single 10 nm-wide filament (arrowhead) and 20–50 nm (arrows and asterisks, respectively) bundles are denoted. (d) Crescentin sample in the presence of 5 mM Mg2þ at pH 7.5. A 20 nm-wide structure is denoted with an arrow. (e) Wild-type crescentin at pH 8.5 in the presence of 5mM Mg2þ. Ten nanometer and wider filaments compose the finely reticu- lated network. molecules per cell, which corresponds to 2.1–4.2 lM (assuming cylindrical cells of 2 lm length and 0.5 lm diameter). Cytoplasmic IF proteins such as vimentin and keratins are characterized by solubility in buffers of high pH and low ionic strength, while lower pH and higher ionic strength strongly favor ﬁlament assembly and hence insol- ubility [Herrmann and Aebi, 2004]. We therefore varied the buffer strength at pH values of 6.5, 7.5, and 8.5 and probed the effects of monovalent Kþ and divalent Mg2þ cations on crescentin assembly. To test the solubility of crescentin under various conditions, we employed an CYTOSKELETON ultracentrifugation-based pelleting assay. The portion of the protein sample found in the pellet was considered as the insoluble fraction. Similar to eukaryotic IF proteins, wild-type crescentin assembly showed a dependence on pH and ionic strength, with lower pH and higher ionic strength strongly favoring assembly (Fig. 4a). Wild-type crescentin was nearly com- pletely soluble at pH 7.5 and 8.5, and assembly at these pH values could only be induced by the presence of 5 mM Mg2þ ions. At pH 6.5, meanwhile, crescentin was highly insoluble except in buffer of very weak ionic strength (5 mM). Monovalent Kþ cations had little effect Analysis of Crescentin Domain Organization 213 n