engleski

Ras proteins regulate actin assembly at endocytic structures via Diaphanous-related formin G

Formins and Arp2/3 complex are major actin assembly factors in eukaryotic cells. While Arp2/3 complex induces branching of the existing filaments, thus creating a dense actin meshwork at the cell's leading edge, formins promote nucleation and elongation of linear actin filaments, mostly in filopodia, stress fibers and cytokinetic rings[1]. Formins are defined by the presence of a highly conserved C-terminal formin homology 2 (FH2) domain preceded by an FH1 domain, which are necessary and sufficient to nucleate actin polymerisation and elongate filaments[2]. Diaphanous-related formins (DRFs) constitute a conserved subfamily in which core FH1 and FH2 domains are flanked by regulatory domains: a GTPase binding domain (GBD), a Diaphanous-inhibitory domain (DID) and a dimerization domain (DD) at the N terminus, and a C-terminal Diaphanous-autoregulatory domain (DAD). DRFs are autoinhibited through interaction between DID and DAD and binding of a Rho GTPase to GBD releases this inhibition. We have characterized formin G (ForG) from the professional phagocyte Dictyostelium discoideum as a Diaphanous-related formin that fully conforms to the canonical domain organization of DRFs, and described its role in large-scale endocytosis[3]. Biochemical assays identified ForG as a rather week actin nucleator that efficiently elongates actin filaments. Live cell imaging demonstrated that ForG was enriched at the plasma membrane during the formation and maturation of macropinocytic cups. Analysis of phagocytosis also revealed localization to nascent phagocytic cups and ForG remained associated with the membrane over the entire course of engulfment and internalization of yeast particle (Fig. 1). Real-time assays of the nutrient uptake showed markedly decreased fluid-phase uptake of TRITC-dextran and phagocytosis of TRITC-labeled yeast particles in forG null cells compared with control cells. Interestingly, yeast-two-hybrid (Y2H) assay failed to identify any interaction of ForG with Rho family GTPases, although DRFs have been previously shown to act exclusively as effectors of Rho GTPases[1]. Strikingly, another Y2H screen and bimolecular fluorescence complementation (BiFC) in vivo identified specifically activated forms of RasB and RasG, but not their dominant negative variants, as ForG interactors (Fig. 2). The role of RasG in macropinocytosis has already been well established. To assess the role of RasB in this process, we have created rasB null cells and tested their nutrient uptake. The uptake of large particles was substantially impaired in rasB deficient cells, while pinocytosis rates were only slightly diminished. Taken together, these data demonstrate the significance of ForG for actin assembly in endocytic structures, with RasB being mainly responsible for its regulation during phagocytosis of large particles, and RasG being the main regulator of ForG in macropinocytosis.

formin ; Arp2/3 complex ; macropinocytosis ; phagocytosis ; Ras

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