| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 607942 | Journal of Colloid and Interface Science | 2013 | 6 Pages |
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 ~2.9 μM. 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.
Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (97 K)Download as PowerPoint slideHighlights► A heterogeneous assay is developed to study gelsolin activity using F-actin. ► Blocking of non-specific binding sites is performed for the reliability of assay. ► The half-maximal coverage of F-actin occurred at 2.9 μM of free Ca2+. ► Severing and capping occurs without addition of Ca2+ after binding to F-actin.
