A network of mixed actin polarity in the leading edge of spreading cells

Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes, such as tissue morphogenesis, cell motility, wound healing, cancer invasion and metastasis. The actin cytoskeletal network is a central driver and regulator of cellular dynamics, that produces membrane protrusions such as lamellipodia and filopodia. In this study, we examined actin organization in the expanding lamellipodia during the early stages of cell spreading. To gain insight into the 3D actin organization, at a molecular resolution, we plated cultured fibroblasts on galectin-8 coated EM grids, an ECM protein presents in disease states. We then combined cryo-electron tomography (cryo-ET) with advanced image processing tools for reconstructing the structure of F-actin in the lamellipodia. This approach enabled us to resolve the polarity and orientation of the filaments, and the structure of the Arp2/3 complexes associated with F-actin branches. We show here that F-actin in lamellipodial protrusions forms a dense network with three distinct sub-domains. One consists primarily of radial filaments, with their barbed ends pointing towards the membrane, the other is enriched with parallel filaments that run between the radial fibers, in addition to an intermediate sub-domain. Surprisingly, a minor, yet significant ([~]10%) population of actin filaments, are oriented with their barbed ends towards the cell center. Our results provide novel structural insights into F-actin assembly and dynamic reorganization in the leading edge of spreading cells.