G-actin diffusion is insufficient to achieve F-actin assembly in fast-treadmilling protrusions.

To generate forces that drive migration of a eukaryotic cell, arrays of actin filaments (F-actin) are assembled at the cell's leading membrane edge. To maintain cell propulsion and respond to dynamic external cues, actin filaments must be disassembled to regenerate the actin monomers (G-actin), and transport of G-actin from sites of disassembly back to the leading edge completes the treadmilling cycle and limits the flux of F-actin assembly. Whether or not molecular diffusion is sufficient for G-actin transport has been a long-standing topic of debate, in part because the dynamic nature of cell motility and migration hinders the estimation of transport parameters. In this work, we applied an experimental system in which cells adopt an approximately constant and symmetrical shape; they cannot migrate but exhibit fast, steady treadmilling in the thin region protruding from the cell. Using fluorescence recovery after photobleaching, we quantified the relative concentrations and corresponding fluxes of F- and G-actin in this system. In conjunction with mathematical modeling, constrained by measured features of each region of interest, this approach revealed that diffusion alone cannot account for the transport of G-actin to the leading edge. Although G-actin diffusion and vectorial transport might vary with position in the protruding region, good agreement with the fluorescence recovery after photobleaching measurements was achieved by a model with constant G-actin diffusivity ∼2 μm/s and anterograde G-actin velocity less than 1 μm/s.