engleski

Dictyostelium discoideum IqgD regulates actin cytoskeleton in large-scale endocytosis

IqgD protein from amoeba Dictyostelium discoideum belongs to an evolutionarily conserved family of IQGAP proteins. IQGAPs are multidomain proteins that act as scaffolds to integrate diverse signalling pathways. They regulate cellular processes that require extensive rearrangement of the actin cytoskeleton, such as migration, adhesion and vesicle trafficking [1]. IQGAPs bind actin filaments directly via their calponin homology domain (CHD) and can further cross-link them into bundles due to their oligomerization. The oligomerization of IQGAPs is facilitated by Rho family GTPases Rac1 and Cdc42, that bind via GAP-related domain (GRD). IQGAPs also regulate actin dynamics via interactions with actin- assembly (Arp2/3, Dia1) and nucleation-promoting (N-WASP) factors responsible for the generation of protrusive structures at the cell leading edge [2]. IqgD is the only Dictyostelium IQGAP that harbours an actin-binding domain. It also contains a coiled-coil region, a GRD and a RasGAP_C- terminal (RGCt) extension, the latter two forming a typical domain architecture of an IQGAP [3]. Using confocal microscopy of live D. discoideum cells we showed that fluorescently labelled IqgD localizes to the entire cell cortex. However, it was significantly enriched at the endocytic cups during macropinocytosis (bulk fluid uptake) and phagocytosis (particle uptake). This localization suggests its involvement in both types of large- scale endocytosis. Hence, we investigated whether IqgD also interacts with small GTPases from the Rho family, in particular those known to be involved in large-scale endocytosis. Using yeast two-hybrid assay we demonstrated that IqgD interacts with Rac1A and Rac1C. Next, we demonstrated that IqgD co-immunoprecipitates with endogenous actin. Finally, we observed that fluorescently labelled IqgD co-localizes with a probe for active Rac1 GTPases and a probe for filamentous (F-) actin in live cells during macropinocytosis, throughout the evolution of a macropinosome. Overall, these results strongly suggest that IqgD acts as a Rho-regulated IQGAP protein that, similar to its mammalian counterparts, regulates actin cytoskeleton remodelling, in particular during the formation of large protrusions in macropinocytosis and phagocytosis.

Dictyostelium ; IqgD ; actin ; large-scale endocytosis

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