Video Imaging of Cofilin-Induced Actin Filament Severing by High Speed AFM

Abstract

Cofilin binds to actin filaments and severs them. Previous electron microscopic studies showed that bound cofilin forms clusters along actin filaments, accompanying supertwisting of the helix (McGough et al., 1997; Galkin et al., 2011). More recent fluorescence microscopic observation revealed that severing of actin filaments occurs at or near the boundary between the cofilin cluster and the bare zone of the filament (Suarez et al., 2012), leading to the hypothesis that structural discontinuity of the filament results in severing. However, the spatial resolution of fluorescence microscopy was insufficient to prove this model unequivocally. To address this and related issues, we attempted to directly image cofilin binding to actin filaments, supertwisting of the helix and subsequent severing of the filaments by high speed atomic force microscopy (HS-AFM), a powerful technique as has been demonstrated previously (Kodera et al., 2010). Skeletal actin filaments in F-buffer, pH 6.8, were allowed to adhere to a mica surface covered fully with lipid membrane doped with 5-10 % of cationic DPTAP lipid. His-tagged human cofilin was then added and HS-AFM was performed. The cationic lipid membrane immobilized actin filaments sufficiently firmly to withstand tapping by the AFM probe, while allowing the filaments to change the helical pitch, so that we were able to observe binding of cofilin to actin filaments as detected by increase in filament height, formation and growth of cofilin clusters, changes of the helical pitch by measuring the crossover length, and subsequent severing of the filament near the boundary of the cofilin cluster. We are currently gathering and analyzing more data, with the aim of establishing the relationship among formation of cofilin clusters, changes in helical pitch and the severing of filaments with a spatiotemporal resolution of several nm and sub-seconds.