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  • Genetic observations from these studies suggest that GMF reg

    2024-06-21

    Genetic observations from these studies suggest that GMF regulates Arp2/3 complex and (S)-(-)-Propranolol hydrochloride dynamics in vivo13, 14. Budding yeast Gmf1 localizes to cortical actin patches, sites of endocytosis assembled by Arp2/3 13, 14. Furthermore, deletion of GMF1 exacerbates the growth defects of a cof1-22 mutant [13], and extends the lifetime of actin patches. In contrast, overexpression of Gmf1 reduces both the number of actin patches and the levels of Arp2/3 complex at actin patches, and rescues specific mutant alleles of the Arp2/3 complex [14]. In addition, gmf1Δ mutants show synthetic defects in actin organization and cell growth with other actin turnover mutants [27]. Collectively, these genetic and biochemical observations provide strong evidence that Gmf1 promotes the remodeling and/or turnover of branched actin arrays in vivo. It is also noteworthy that gmf1Δ cells exhibit disorganized actin cables (actin structures polymerized by formins) [27], and defects in mitochondrial distribution likely caused by disorganized cables, as suggested by its alias name, Aim7, Altered inheritance of mitochondria 7 [28]. Importantly, the cable defects of gmf1Δ mutants may arise from reduced actin patch turnover, leading to an imbalance in the homeostatic distribution of actin subunits between patches and cables [29].
    Mechanism of GMF-Induced Debranching Mechanistic work has revealed that GMF debranches actin filaments by a mechanism related to cofilin severing of actin filaments, except that it is specialized for Arp2/3–actin junctions 13, 26, 30. It has been reported that there are two binding sites for Gmf1 on the Arp2/3 complex, one high affinity and one low affinity (with Kd=10nM and 1μM, respectively) [26]. Furthermore, single particle electron microscopy showed that Gmf1 induces an open/inactive conformation in the Arp2/3 complex [31], related to the effects of coronin on Arp2/3 structure 32, 33. Employing fluorescence anisotropy, mutagenesis, and chemical crosslinking, it was found that Arp2/3 complex-binding is mediated by a broad surface on Gmf1 (‘Site 1’), analogous to the globular/filamentous (G/F) actin-binding surface on cofilin (Figure 1B,C) [26], and suggests that Site 2 (analogous to the F-site on cofilin) interacts with the first conventional actin subunit in the daughter filament. Both Site 1 and Site 2 were found to be important for Gmf1 functions in yeast cells, suggesting that debranching is critical for Gmf1 function in vivo. In parallel, when the crystal structure of GMFγ bound to mammalian Arp2/3 complex was solved [30], it validated one of the two proposed binding sites of GMF on the Arp2/3 complex, and provided atomic-level detail of the interaction. This structure reveals that GMF directly contacts the Arp2 and p40/ARPC1 subunits, which is presumed to represent the high-affinity binding site for GMF on the Arp2/3 complex, and suggests that surfaces on Arp2 may have coevolved with GMF to maintain GMF interactions while excluding cofilin interactions [30]. A recent study using molecular dynamics simulations provides additional support for the existence of a second, lower affinity binding site for GMF on the Arp2/3 complex, that involves interactions with the Arp3 subunit [34]. The above-mentioned studies also established that GMF catalyzes branch dissociation using its two distinct functional surfaces, with Site 1 binding to the Arp2 and p40/ARPC1 subunits and Site 2 binding to the first actin subunit in the daughter filament. Site 1 on GMF competes for binding with the nucleation promoting factor (NPF) WASp-VCA domain, explaining how GMF attenuates VCA-induced Arp2/3-mediated nucleation [30] (Figure 2C). Because GMF and cofilin are structural homologues with similarly positioned functional surfaces/sites (Figure 1B), GMF may destabilize Arp2/3–actin branch junctions by a mechanism related to how cofilin destabilizes actin filaments to induce fragmentation. This argument is strengthened by GMF and cofilin both preferring the ADP-bound state of their ligands, Arp2/3 complex, or actin, respectively. Given that cofilin induces a twist in F-actin conformation and alters actin–actin contacts 35, 36, it is possible that GMF exerts its debranching effects by similarly inducing conformational changes at Arp2/3–actin junctions.