Understanding actin turnover – no longer a mere speckle on the horizon

The lamellipodia that push forward the leading edge of motile cells contain a dynamic network of actin filaments. These movements require recruitment of molecules for polymerization of actin filaments and their subsequent disassembly. The Arp2/3 complex plays a key role in actin polymerization, promoting nucleation and dendritic organization of filaments. Depolymerization is thought to be achieved by proteins of the ADF/cofilin family. Interest currently focuses on how actin is dynamically regulated (reviewed in Ref. [1xThe lamellipodium: where motility begins. Small, J.V et al. Trends Cell Biol. 2002; 12: 112–120Abstract | Full Text | Full Text PDF | PubMed | Scopus (551)See all References[1]). Watanabe and Mitchison now report a new approach for analyzing spatial and temporal regulation of actin polymerization and depolymerization in vivo [2xSingle-molecule speckle analysis of actin filament turnover in lamellipodia. Watanabe, N and Mitchison, T.J. Science. 2002; 295: 1083–1086Crossref | PubMed | Scopus (260)See all References[2].β-Actin fused to green-fluorescent protein (GFP) expressed in fibroblasts was incorporated at very low concentrations such as to form speckles on actin filaments, each speckle representing a single GFP–actin molecule. By observing speckles, Watanabe and Mitchison analyzed actin depolymerization. First, in order to measure rapid changes in overall depolymerization rates within a single cell, a set of speckles was identified in one reference image, and the decrease in their numbers was followed over subsequent images. The half-life of filaments in lamellipodia averaged 30 seconds. Second, to measure the lifetime distribution of actin filaments, speckles were monitored from their emergence to their disappearance. The average filament lifetime was almost uniform across the lamellipodium, except for a 1.6-fold increase at the most distal edge, referred to by the authors as the tip region. The distribution of polymerization activity was determined by following the appearance of speckles, and comparing this with the steady-state filament distribution. The results revealed that there are two distinct regions of actin polymerization activity, with a region within 1 μm of the tip showing rates of new speckle formation higher than those in the rest of the lamellipodia.By using this single-molecule analysis, the authors demonstrated that, in the lamellipodium body, basal polymerization and depolymerization occur with constant kinetics. In addition, they modeled two polymerization mechanisms. This method should help to clarify the regulation of basal polymerization compared with that occurring at the tip, which is governed by physical restriction of the plasma membrane and promoted by factors such as Scar and VASP/Mena proteins.