Abstract
Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the background of the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy. Our work identifies the first three gelsolin homology domains (1–3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4–6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (∼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼ µM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. Flightless-I was found to interact with actin and affect actin dynamics in a calcium-independent fashion in vitro, suggesting the lack of calcium-mediated activation and conformational change of protein. For the comparative structural analysis of the six gelsolin homology domains (GH16) of gelsolin and Flightless-I, we used a combination of biophysical and biochemical approaches. The use of both internal (tryptophans) and external (8-anilinonaphthalene-1-sulfonic acid; ANS) fluorophores revealed that calcium-binding induces structural changes in gelsolin but the conformational behavior of Flightless-I GH16 was not significantly affected by the divalent cation. Different kinetics of limited proteolysis observed for gelsolin and Flightless-I GH16 further strengthened this conclusion. Our experimental findings are supported by bioinformatics analysis predicting that the sequence elements responsible for Ca2+-activation of GSN are not conserved in Flightless-I GH16.
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