Relationship between Arp2/3 complex and the barbed ends of actin filamentsat the leading edge of carcinoma cells after epidermal growth factor stimulation

Using both light and high resolution electron microscopy, we analyzed the s patial and temporal relationships between the Arp2/3 complex and the nuclea tion activity that is required for lamellipod extension in mammary carcinom a cells after epidermal growth factor stimulation. A rapid two- to fourfold increase in filament barbed end number occurs transiently after stimulatio n and remains confined almost exclusively to the extreme outer edge of the extending lamellipod (within 100-200 nm of the plasma membrane). This is ac companied by an increase in filament density at the leading edge and a gene ral decrease in filament length, with a specific loss of long filaments. Co ncomitantly, the Arp2/3 complex is recruited with a 1.5-fold increase throu ghout the entire cortical filament network extending 1-1.5 mu m in depth fr om the membrane at the leading edge. The recruitment of the Arp2/3 complex at the membrane of the extending lamellipod indicates that Arp2/3 may be in volved in initial generation of growing filaments. However, only a small su bset of the complex present in the cortical network colocalizes near free b arbed ends. This suggests that the 100-200-nm submembraneous compartment at the leading edge of the extending lamellipod constitutes a special biochem ical microenvironment that favors the generation and maintenance of free ba rbed ends, possibly through the locally active Arp2/3 complex, severing or decreasing the on-rate of capping protein. Our results are inconsistent wit h the hypothesis suggesting uncapping is the dominant mechanism responsible for the generation of nucleation activity. However, they support the hypot hesis of an Arp2/3-mediated capture of actin oligomers that formed close to the membrane by other mechanisms such as severing. They also support point ed-end capping by the Arp2/3 complex, accounting for its wide distribution at the leading edge.