Analysis of the regulation of actin cytoskeleton dynamics using Dronpa, a photochromic fluorescent protein

Cofilin stimulates actin filament disassembly by severing and depolymerizing actin filaments and accelerates actin filament turnover. It is unclear whether cofilin contributes to stimulus-induced actin filament assembly by supplying actin monomers for polymerization or by creating free barbed ends through its severing activity. By measuring the time-lapse fluorescence decay of photoactivated Dronpa-actin, we have assessed the cytoplasmic actin monomer pool in living cells and provide evidence that cofilin is involved in production of more than half of the actin monomers in the cytoplasm. Actin monomers in the cytoplasm were incorporated into the tip of the lamellipodium, and incorporation depended both on cofilin activity and on the size of the cytoplasmic actin monomer pool. We therefore propose that cofilin critically contributes to stimulus-induced actin filament assembly and lamellipodium formation by supplying actin monomers abundantly to the cytoplasm.