Arp2/3 complex‐mediated actin polymerisation occurs on specific pre‐existing networks in cells and requires spatial restriction to sustain functional lamellipod extension

The classical Arp2/3‐mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3‐mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex‐mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3‐mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3‐mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3‐mediated networks are intrinsically three‐dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre‐existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex‐mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex‐mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network. Cell Motil. Cytoskeleton 2006. © 2006 Wiley‐Liss, Inc.