A visualized observation of calcium-dependent gelsolin activity upon the surface coverage of fluorescent-tagged actin filaments

Gelsolin regulates the dynamics of F-actin by binding to F-actin to sever and cap. In the present study, a novel approach is introduced to observe gelsolin activity through the coverage of surface-bound F-actin. Gelsolin was immobilized on streptavidin coated surface using biotinylation and, as a result, the interaction between gelsolin and F-actin was visualized. Consequently, the coverage of F-actin reflects the activity of gelsolin as a function of free Ca2+ concentrations. In order to prevent non-specific binding of F-actin, the combinations of BSA and Tween-20 as blocking agents were investigated. Moreover, the measurement of the length of F-actin with actin-gelsolin mixtures at various ratios provided the verification of gelsolin activity after biotinylation. The data shows the increase in Ca2+ concentration leads to a proportional increase in F-actin coverage, giving to half-maximal coverage at similar to 2.9 mu M. Furthermore, the length of bound F-actin was found to decrease along with increasing Ca2+ concentration, and full-length F-actin was rarely observed. This may suggest that severing and capping activities of gelsolin occur without more additional Ca2+ for subsequent activation after full-length gelsolin binds to a side of F-actin. This finding may provide a key to understand gelsolin activity. (C) 2012 Elsevier Inc. All rights reserved.