IqgD is a Rac1-interacting IQGAP required for efficient growth of Dictyostelium discoideum on bacterial lawns.

Abstract

Background: Phagocytosis of surface-bound microbes is essential for host defence and environmental feeding, yet the mechanism by which macrophages remove surface-bound particles has only recently been described. This process involves the formation of an F-actin-rich, force-bearing ring around the surface-attached particle. Here, we identify the IQGAP-related protein IqgD from the professional phagocyte Dictyostelium discoideum as a key regulator of mechanically demanding phagocytosis. IQGAPs are large multidomain scaffold proteins that interact with Rho family GTPases and F-actin. IqgD contains a calponin homology domain (CHD), a GAP-related domain (GRD), a RasGAP C-terminal (RGCT), and an extreme C-terminal (CT) domain.

Methods: In this study, we used biochemical and imaging approaches with full-length and truncated protein variants to investigate whether IqgD interacts with D. discoideum Rho GTPases and F-actin. We also performed comprehensive phenotypic characterisation of IqgD-deficient cells to determine the cellular function of IqgD.

Results: We show that the CHD is essential for F-actin binding and cortical localisation, while the GRD and CT domains mediate interactions with Rac1 GTPases and the actin-bundling proteins cortexillins. Moreover, similar to mammalian IQGAPs, IqgD maintains Rac1 in its active conformation. Although IqgD is enriched in macropinocytic and phagocytic cups, it is not required for fluid uptake or internalisation of bacteria from suspension. However, loss of IqgD markedly reduces growth on bacterial lawns and strongly impairs uptake of surface-attached microbeads and yeast particles. Furthermore, IqgD localises to F-actin-rich ring-like structures that form around surface-bound particles at the basal cell surface.

Conclusions: IqgD is not required for all forms of macroendocytosis; rather, it is specifically required for mechanically demanding phagocytosis. This includes the formation of enlarged phagocytic cups during phagocytosis of yeast particles or the generation of greater force, as in phagocytosis of surface-bound particles and bacteria within bacterial lawns. Similar to phagocytosis of surface-attached particles in mammalian macrophages, D. discoideum also forms an F-actin-rich ring around the particle at the cell base, suggesting that force-driven particle detachment and internalisation may be an evolutionarily conserved mode of substrate-dependent phagocytosis. Our findings provide mechanistic insight linking IqgD with Rac1, cortexillins, and F-actin in the regulation of demanding forms of phagocytosis.