FIGURE 2.

Actin binding and bundling activities of wild type and mutant fascin. A, results of binding (high speed; 224,000 × g) and bundling (low speed; 10,000 × g) co-sedimentation (pelleting) assays with filamentous F-actin, including control experiments (actin and fascin alone). Supernatant (S) and pellet (P) fractions were analyzed by gel electrophoresis. Also shown is a description of the mutants, including specific amino acids substituted in each mutant and schematic representations of their locations in the structure (β-trefoil domains colored according to Fig. 1; mutated residues and Ser³⁹ represented by blue balls and a red ball, respectively). Mutants are named according to the numbers of the β-trefoil domains that were mutated in each case. B, representative transmission electron micrographs of negatively stained actin filaments alone and in the presence of wild type or mutant fascin (aligned with A). Low magnification images (left; scale bar, 200 nm) show that nearly all the actin filaments are incorporated into bundles in the presence of wild type fascin or fascin mutants. High magnification images (right; scale bar, 50 nm) show examples of a thick ordered bundle with periodic striations (indicated by arrows) formed by wild type fascin and thinner bundles with aberrant morphology formed by the fascin mutants. The bundles formed by mutants 2-3 and 3-4 often show striations (indicated by arrows). C, quantitative analysis of the bundles formed in the presence of wild type and mutant fascin (n = 10). Asterisks indicate statistically significant differences relative to wild type fascin (p < 0.02). Error bars, S.D.