Figure 6.

The CARD9–CARD forms filaments in a Zn²⁺-regulated manner that are capable of directly templating the Bcl10 helical assembly. A, CARD9–CARD filaments, visualized by NS-EM. Filaments were generated as in C, red line, with NS-EM samples prepared at 90 min. B, NS-EM micrograph of 200 μm CARD9–CARD saturated with 1:1 Zn²⁺, sample taken just before EDTA addition in C. C, CARD9–CARD filament assembly, monitored by absorbance at 350 nm. All samples contain CARD9–CARD saturated with 1:1 Zn²⁺ and 150 mm NaCl (except as indicated); at t = 0, 250 μm EDTA was added to the indicated samples. At 90 min, 250 μm ZnCl₂ was added to all samples. Error bars represent the standard deviation of three technical replicates. D, left, 20-Å resolution NS-EM reconstruction of the CARD9–CARD helical assembly; right, 20-Å resolution NS-EM–derived Bcl10–CARD helical assembly solved by Qiao et al. (23) (EMD-5729). The dominant 3-, 4-, and 7-start helical symmetries are indicated, highlighting the similarity between the structures. E, fluorescence polarization assay utilizing 2 μm MBP–Bcl10 sparsely tagged with Alexa Fluor 488 dye. At time t = 0, TEV protease, CARD9–CARD monomer, and/or CARD9–CARD filaments were added as indicated. Error bars represent the standard deviation of three technical replicates. F–H, NS-EM micrograph of Bcl10 filaments alone (F), CARD9–CARD filaments alone (G), or CARD9–CARD filament-nucleated Bcl10 filaments (H) generated under identical conditions as in E (5:1 ratio, red), 2 min after addition of TEV protease. H, white arrows indicate CARD9–CARD-to-Bcl10 transitions.