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 Ca(2+) 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 Ca(2+) 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 Ca(2+) concentration, and full-length F-actin was rarely observed. This may suggest that severing and capping activities of gelsolin occur without more additional Ca(2+) for subsequent activation after full-length gelsolin binds to a side of F-actin. This finding may provide a key to understand gelsolin activity.
Lane Department of Computer Science and Electrical Engineering, West Virginia University, Morgantown, WV 26506, USA.
Yongkuk
Y
Lee
Ming-Yuan
MY
Wei
Parviz
P
Famouri
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D019921:Biotinylation; D002118:Calcium / Q000378:metabolism*; D018260:Gelsolin / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D018260:Gelsolin; D002118:Calcium