engleski

The RasGAP protein IqgC regulates cell-substratum adhesion in ameboid cells

Proper cell-substratum adhesion is crucial for motility, phagocytosis and cytokinesis of single cells, and is also essential for complex biological processes such as embryonic development, wound healing, and the maintenance of tissue integrity. Amoeba Dictyostelium discoideum is a unicellular model organism that possesses homologs of many proteins involved in cell- substratum adhesion in higher eukaryotes, for instance, talin, paxillin, and small GTPase RapA. IqgC is a RasGAP protein that inactivates small GTPase RasG and negatively regulates phagocytosis and macropinocytosis. We observed that cells lacking IqgC have impaired attachment to the substratum. Indeed, when the detachment of cells from the bottom of petri dishes was assessed using a shaking assay, iqgc-null cells were more easily detached than wild-type cells, and the expression of recombinant IqgC in mutant cells rescued this defect. We also observed the localization of fluorescently tagged IqgC to the punctiform adhesion structures on the ventral part of moving cells using TIRF microscopy. To determine which protein domain is important for the localization and function of IqgC in adhesion, truncated variants of the protein were expressed in mutant cells. IqgC consists of two main functional domains – a GRD (GAP-related domain) and an RGCt (RasGAP C-terminal domain). The GRD domain binds small GTPases in homologous proteins, for example in human IQGAP1, while the RGCt domain is important for binding PtdIns(4, 5)P2, E- cadherin and β-catenin. In order to compare the localization of these two domains with the fulllength protein, the iqgc-null cells were transfected with the fluorescently labeled YFP- IqgC, YFP-GRD and YFP-RGCt constructs. YFP-IqgC and YFP-RGCt localized to the ventral punctiform adhesion structures, while YFP-GRD did not. Furthermore, we tested the ability of these constructs to rescue the adhesion defect of iqgc- null cells. Only YFP-IqgC, and none of the truncated constructs, rescued the impaired adhesion. Therefore, we conclude that the RGCt domain is sufficient for the localization of IqgC to the adhesion structures, but it needs additional parts of the full-length protein (possibly the GRD domain) to fully exert its role in the cell-substratum adhesion.

adhesion ; Dictyostelium ; IqgC

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