Fascin structural plasticity mediates flexible actin bundle construction.

Fascin crosslinks actin filaments (F-actin) into bundles that support tubular membrane protrusions including filopodia, stereocilia, and microvilli. Fascin dysregulation drives aberrant cell migration during metastasis, and fascin inhibitors are under development as cancer therapeutics. Here, we use cryo-electron microscopy and cryo-electron tomography coupled with custom denoising to probe fascin's F-actin crosslinking mechanisms across spatial scales. Our fascin crossbridge structure reveals an asymmetric F-actin binding conformation that is allosterically blocked by the inhibitor G2. Reconstructions of seven-filament hexagonal bundle elements and variability analysis show how structural plasticity enables fascin to bridge varied inter-filament orientations, accommodating mismatches between F-actin's helical symmetry and bundle hexagonal packing. Tomography of many-filament bundles uncovers geometric rules underlying emergent fascin binding patterns, as well as the accumulation of unfavorable crosslinks that limit bundle size. Collectively, this work shows how fascin harnesses fine-tuned nanoscale structural dynamics to build and regulate micron-scale F-actin bundles.