Spatial high resolution of actin filament organization by PeakForce atomic force microscopy

OBJECTIVES: To investigate the heterogeneous feature of actin filaments (ACFs) associated with the cellular membrane in HeLa and HCT‐116 cells at the nanoscale level. MATERIALS AND METHODS: Fluorescence microscopy coupled with atomic force microscopy (AFM) was used to identify and characterize ACFs of cells. The distribution of ACFs was detected by Fluor‐488‐phalloidin–labelled actin. The morphology of the ACFs was probed by AFM images. The spatial correlation of the microvilli and ACFs was explored with different forces of AFM loading on cells. RESULTS: Intricate but ordered structures of the actin cytoskeletons associated with cellular membrane were characterized and revealed. Two different layers of ACFs with distinct structural organizations were directly observed in HCT‐116 and HeLa cells. Bundle‐shaped ACFs protruding the cellular membrane forming the microvilli, and the network ACFs underneath the cellular membrane were resolved with high resolution under near‐physiological conditions. Approximately 14 nm lateral resolution was achieved when imaging single ACF beneath the cellular membrane. On the basis of the observed spatial distribution of the ultrastructure of the ACF organization, a model for this organization of ACFs was proposed. CONCLUSIONS: We revealed the two layers of the ACF organization in Hela and HCT‐116 cells. The resolved heterogeneous structures at the nanoscale level provide a spatial view of the ACFs, which would contribute to the understanding of the essential biological functions of the actin cytoskeleton.