MLCK and N75 structure and interactions with actin-containing structures. (A) Cartoon showing MLCK structural elements including the region corresponding to N75. Domains are labeled, and the functions are shown below. For more information, see Hong et al. (2011). (B–G) Merged images of QD-MLCK and QD-N75 (green) colocalized on TRITC-labeled actin (red). MLCK (B–D) and N75 (E–G) on MLCK-induced actin bundles (B and E), single actin filaments (C and F), and HASMC stress fibers (D and G). (B) 300 nM actin was applied and washed, and bundling was induced by adding 200 nM MLCK for 12 min (similar to Fig. S1 F). Actin bundles (brighter) and apparent single actin filaments (dimmer, long arrows) are shown. QD-MLCK (2 nM of biotinylated MLCK, 8 nM QD; Method A) was applied for 30 min, followed by a wash. Short arrows indicate QD-MLCK colocalized on actin bundles, and arrowhead indicates QD-MLCK colocalized on single actin filament. (C) 100 nM 5% biotinylated actin (red) applied to a PEG surface and washed, and then QD-MLCK was applied as in B. Most actin is not bundled. The arrow shows one QD colocalized. (D) MLCK is bound to HASMC stress fibers. (E) Same as B, except with 1 µM N75 and 0.2 nM QD. (F) Single actin filaments were bound to a poly-lysine–coated coverslip followed by 1 µM N75 and 0.2 nM QD. QD-N75 colocalizes to actin single filaments. (G) Same as D, except with N75. Bar shown is the same for all images, which are 512 pixels² (54 µm²).