期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：47
页码：E7464-E7473
DOI：10.1073/pnas.1611024113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMacropinocytosis and phagocytosis are two Ras-regulated, highly related processes of great physiological relevance collectively termed large-scale endocytosis. Both are actin-driven and entail engulfment of extracellular material by crown-like protrusions. Aside from the Arp2/3 complex, which serves as the main nucleator of branched actin filaments at the cup rim, the underlying mechanisms of actin assembly still remain elusive. Here, we analyzed the role of Diaphanous-related formin G (ForG) from Dictyostelium by biochemical, genetic, and imaging techniques. Our data demonstrate that this formin exhibits a rather weak nucleation activity and imply that ForG-mediated filament elongation synergizes with the Arp2/3 complex in actin assembly. Finally, we identify ForG as a Ras-regulated formin and show its significance for actin assembly in endocytic structures. Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.
