br GMF Remodels Actin Networks at

GMF Remodels Actin Networks at the Leading Edge How are the conserved activities of GMF used in vivo to regulate branched GSK1210151A networks (e.g., at sites of endocytosis and at the leading edge) (Figure 2A,B)? In animal cells, the regulatory effects of GMF on actin networks appear to govern lamellipodial dynamics and cell motility (Box 1). In IA32 mouse embryonic fibroblasts, GMFβ localizes to mature rather than nascent lamellipodia, and overexpression of GMFβ demonstrates that it promotes lamellipodial retraction and ruffling, while depletion of GMFβ dramatically alters leading-edge dynamics [10]. Consistent with observations made at yeast cortical actin patches [14], knockdown of GMFβ leads to increased Arp2/3 levels in lamellipodia. Conversely, GMFβ overexpression reduces Arp2/3 levels, suggesting that GMFβ catalyzes Arp2/3 turnover in actin networks [10]. Further, a point mutant was introduced into GMFβ to impair debranching, but not nucleation inhibition (designed based on the ‘Site 2’ mutant in yeast Gmf1; [26]). Experiments using this mutant and a small molecule inhibitor of the Arp2/3 complex demonstrated that GMFβ debranching activity is critical for proper leading-edge dynamics [10]. Thus, GMF debranching activity is essential for its in vivo functions in promoting actin network remodeling and turnover. However, no tool has yet been generated to disrupt GMF inhibition of Arp2/3-mediated nucleation without also disrupting debranching, making it more difficult to assess the importance of the nucleation inhibition activity of GMF in vivo. Additional support for the regulation of lamellipodial dynamics by GMF comes from studies in Drosophila S2 cells and embryonic cells undergoing collective migration [25]. Here, GMF is enriched in lamellipodia undergoing retraction, a phase in which branched actin networks are rapidly disassembled and/or remodeled (debranched) into parallel bundles called ‘arcs’ (Figure 2B) [37]. GMF mutant flies also have reduced rates of border cell migration [25], demonstrating the importance of GMF function for directed cell migration in the physiological context of an intact animal. Similar roles may also explain the functions of the GMFγ isoform, which is highly expressed in immune tissues 8, 38. GMFγ has important roles in hematopoietic lineage differentiation 9, 39, 40, 31, lamellipodial dynamics and chemotaxis in neutrophils and T cells 41, 42, and vascular development in zebrafish [7]. The discovery that GMF drives lamellipodial retraction rather than protrusion lends important insights into how GMF promotes cell motility. Specifically, GMF may help remodel branched actin networks into unbranched structures such as actin arcs [37], which form parallel to the leading edge and treadmill rearwards [42]. Additionally, GMF may promote motility by governing the density of branches in actin networks at the leading edge. A number of studies, reviewed in more detail elsewhere [43], have explored the link between branch density, lamellipodial dynamics, and cell migration by altering expression levels of actin elongation factors (e.g., ENA/VASP, formins, and CARMIL), inhibitors of elongation (e.g., capping protein), branch stabilizers (e.g., cortactin), and branch destabilizers (e.g., coronin). Together, these studies suggest that densely branched networks comprised of shorter filaments lead to slow and steady lamellipodial protrusion, and there is an inverse correlation between protrusion rate and persistence [43]. Consistent with this view, depleting GMF, which should increase branch density, increases protrusion persistence [10]. In addition to these roles, GMF debranching is predicted to expose minus ends of filaments, thereby increasing the depolymerization flux and recycling of the Arp2/3 complex, which in turn allows for continued network assembly at the leading edge [44]. Persistently protruding lamellipodia drive more continual directional cell migration 43, 45, and studies on GMF support this [10]. However, there are conflicting reports about the relationship between lamellipodial protrusion rates and cell speed. In one study, slow and steady protrusion correlated with faster cell speed [46], while in a different study, rapid protrusion correlated with faster cell speed [10]. It is possible that other parameters, such as the frequency and extent of lamellipodial retraction events, rather than protrusion rate, are the more relevant determinants of cell speed [45]. Finally, GMF may suppress unproductive lateral cell protrusions, leaving more Arp2/3 complex and actin monomers available for advancement of the leading edge of the cell 10, 25, 41. This idea is similar to the proposed mechanism for Arpin, another direct inhibitor of the Arp2/3 complex that steers cell motility [47]. Indeed, in some cell types, the Arp2/3 complex is dispensable for cell migration, yet required for steering cells toward haptotactic [48] or chemotactic signals [49].