Using Corralled Diffusion of Microspheres to Probe the Mesh Size of Actin Filament Network*

Actin is an important cytoskeletal filament that helps maintain cell shape and drive cell movement and division. Whereas the polymerization and function of individual actin filaments are well-understood, key properties of the actin filament network remain to be elucidated. In particular, the typical spacing between actin filaments in a network ("mesh size") was recently shown to hinder the delivery of larger DNA molecules from reaching the cell’s nucleus, which can have important implications in non-viral gene delivery and expression. Here we employ the "corralled diffusion" of sub-micron-sized spheres to probe the mesh size of the actin filament network in vitro. We varied the concentration of actin monomers to controllably tune the mesh size in a network. We used single-particle tracking to determine the trajectory of individual beads diffusing in the network. We then evaluated the mean square displacement of the bead trajectory for each actin concentration tested.  We found that the Brownian diffusion of individual beads is partially confined by the actin network ("corralled diffusion"), and that the degree of this partial confinement correlates inversely with actin concentration employed. Together our result suggests corralled diffusion of microspheres as a potential and passive probe to understand the mesh size of actin filament network. Findings may be important for understanding how actin networks impact the diffusion and the spatial mobility of important proteins and/or DNA in cells.