Actin Filaments Bundling Mechanisms by Fascin in Filopodia were Revealed with Cryo-ET

Abstract

Filopodia are finger-like protrusions at the leading edge of migrating cells and are crucial for cell motilities as antennae. It is known that actin filaments are bundled hexagonally and provide rigidity to filopodia by fascin, which is an actin filament bundling protein and plays a central role of actin bundling. However, molecular mechanisms of their formation have been still unclear. We, here, observed the filopodia of intact whole cells fixed by rapid freezing and revealed their three-dimensional maps by cryo-electron tomography and image processing; the actin filaments’ bundling structure was clarified at high resolutions under close-to-life conditions. It is found that the actin filaments in vivo were more numerous than those in the bundles reconstructed in vitro, whereas actin filaments and fascin possess dynamic properties in filopodia. In additions, each actin filament in the filopodia showed a slightly loose twist similar to those bundled by fascin in vitro. The cross-linking structures between actin filaments were also observed clearly and were determined to be fascin by comparison with actin and fascin atomic models. Then we identified three actin binding sites of fascin against two adjacent actin filaments: one bound fascin in two binding regions with wide surfaces and the other did in a tiny region. We propose that these two different binding manners between actin and fascin should provide rigid bundles but flexible and dynamic performance.