Dissecting the role of formins in large-scale endocytosis

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Phagocytosis and macropinocytosis are closely related and evolutionary conserved clathrin-independent endocytic processes. Both are actin-driven processes downstream of Ras and Rac signaling that entail rearrangements of the plasma membrane to engulf extracellular material followed by delivery of the ingested material into lysosomes for extraction of nutrients. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis still remain elusive. This project aims to clarify the specific contribution of formin family members to both macropinocytosis and phagocytosis, and elucidate their prospective synergy to each other and with the Arp2/3 complex in actin assembly during large-scale endocytosis downstream of Ras and Rac signalling. In previously published work, we established that the Diaphanous-related formin ForG is regulated by RasB and RasG and that it exhibits rather poor nucleation but effective actin-filament elongation activities. ForG contributes to the generation of actin filaments localized at the cup base and its activity is required for the generation of about 50% of the actin filaments in this structure. Since the Arp2/3 complex is apparently activated only at the expanding rims of the cups, it remains currently unclear which factors are at play that are responsible for the synthesis of the other half of the actin filaments in the base as well as for the filaments closer to the rim. Previous studies with fluorescently tagged biosensors in fact revealed that the entire periphery is decorated with active Rac, suggesting the presence of other factors than the Rac-binding Scar/WAVE complex, which appears to be required activating Arp2/3 complex in the tips. Of note, with ForB we have recently identified a second Diaphanous-related formin that prominently accumulates in its active form at the entire periphery of macropinosomes. Since this type of formins is in most cases activated by Rho-family GTPase members and because all other Dictyostelium formins that are expressed during the growth phase can be functionally excluded, we hypothesize that ForB could be the missing factor which is required together with ForG for the assembly of the majority of actin filaments in endocytic cups. The dissection of formin function in large-scale endocytosis is undoubtedly a challenging task, but we anticipate that by using various complementary approaches including thorough biochemical analysis of ForB, the identification of its regulatory Rac protein as well as the comprehensive cell biological analyses of single- and double-knockout cell lines as specified in the work programme will finally reveal the molecular activities and mechanisms underlying regulation of actin assembly in these large endocytic structures.