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    • estrone In pioneering work from Ikeda and colleagues showed

    2023-10-01

    In 2006, pioneering work from Ikeda and colleagues showed that GMF localizes to and regulates the estrone cytoskeleton (see Glossary), and noted its sequence similarity to ADF/cofilin[11]. In 2009, the structure of GMF [6] was solved, revealing it to be a bona fide member of the actin depolymerizing factor homology (ADF-H) family, which includes: ADF/cofilin, Twinfilin, actin binding protein 1 (Abp1)/Drebrin, and Coactosin 6, 12. All known ADF-H proteins bind to either actin and/or actin-related proteins (Arps), and have conserved roles in actin cytoskeleton remodeling[12]. Below, we highlight recent work defining the biochemical and cellular roles of GMF in binding the Arp2/3 complex to remodel branched actin filament networks and thereby control endocytosis and cell motility. Finally, we attempt to integrate these new functions with its earlier assignment as a cell signaling and differentiation factor.
    GMF Branches Out as an Actin Regulator GMFγ was first established as a component of the actin cytoskeleton when it was shown to be highly expressed in microvascular endothelial cells and found to colocalize with actin at the leading edge of migrating cells [11]. It was also established that GMFγ has 39% similarity in sequence to ADF/cofilin, a prominent actin filament-severing protein, and that GMFγ coimmunoprecipitates with the actin-nucleating Arp2/3 complex. Shortly thereafter, the NMR structures of GMFβ and GMFγ were solved, and found to be remarkably similar to ADF/cofilin [6] (Figure 1D,E). These pioneering studies prompted other groups to further investigate the cytoskeletal functions of GMF in other model systems. In 2010, two studies in yeast revealed exciting new functions for the Saccharomyces cerevisiae and Saccharomyces pombe homologs of GMF, both called Gmf1 (Figure 2) 13, 14. These studies showed that purified Gmf1 lacks appreciable affinity for G-actin or F-actin, but directly regulates activities of the Arp2/3 complex. The Arp2/3 complex contains two Arps, Arp2 and Arp3, and upon binding to an activator such as WASp or WAVE, it binds to the side of an existing actin ‘mother filament’ and nucleates formation of a ‘daughter filament’ branch (Box 1). In this manner, the Arp2/3 complex forms arborized or ‘dendritic’ actin filament networks, including those found at the leading edge of migrating cells and at sites of endocytosis. Gmf1 was found to inhibit actin nucleation co-stimulated by the Arp2/3 complex and the verprolin/cofilin/acidic (VCA) domain of WASp, as demonstrated both in bulk actin assembly assays and single-filament total internal reflection fluorescence (TIRF) microscopy assays 13, 14. Inhibition of the Arp2/3 complex involved competition between Gmf1 and the WASp VCA domain for binding the Arp2/3 complex, and strong inhibition required low micromolar concentrations of Gmf1. Gmf1 also catalyzed debranching, or ‘pruning’ of daughter filaments by dissociation at branch sites [13]. Earlier, it had been shown that branch junctions generated by the Arp2/3 complex in vitro are unexpectedly stable, failing to dissociate for tens of minutes in the absence of additional factors 15, 16. In contrast, branches in vivo at the leading edge and at endocytic sites turn over in seconds 17, 18, suggesting the existence of cellular factors that might catalyze debranching. Low nanomolar concentrations of Gmf1 induced rapid ‘pruning’ of daughter filaments from mother filaments without severing elsewhere along the filaments [13]. The much higher concentration of GMF required for inhibition of nucleation by the Arp2/3 complex versus debranching may reflect a requirement for GMF to occupy two separate binding sites on the Arp2/3 complex to block nucleation, one high affinity and one low affinity site (see below). These observations established GMF as a dedicated debranching factor and suggested that it may be involved in remodeling branched actin networks into unbranched arrays, as has been observed to occur only a short distance from the leading edge 19, 20, 21. Other factors such as ADF/cofilin and coronin stimulate debranching in vitro22, 23, but additionally promote severing of actin filaments along their lengths 13, 24, and may therefore lead to actin network disassembly rather than remodeling, (i.e., they may promote the dissolution of filaments to yield actin monomers, as opposed to the rearrangement of filaments within a network). Subsequent studies showed that the two biochemical functions of GMF on the Arp2/3 complex (inhibition of nucleation and stimulation of debranching) are conserved for the Drosophila homolog and both mammalian isoforms of GMF (14, 25, 26 M.O. Sweeney, PhD Thesis, Brandeis University, 2014). Thus, GMF activities on the Arp2/3 complex are conserved across great evolutionary distances, consistent with the high degree of sequence conservation in the two key functional surfaces of GMF (Figure 1B).