engleski

A novel function of the RasGAP protein IqgC in cell adhesion

Proper cell adhesion is crucial for many cellular processes, such as cell migration, phagocytosis and differentiation. In the migrating Dictyostelium discoideum cells, transient stationary adhesion foci form on the ventral cell surface underneath the cell body and the pseudopods. These adhesion foci can be discerned from the F-actin-containing puncta presumably involved in transmitting the traction forces to the substratum. The adhesion foci contain a number of proteins homologous to those involved in adhesion in higher eukaryotes, such as talin, paxillin, Rap1, and others. IqgC is a RasGAP protein, which inactivates small GTPase RasG in D. discoideum by stimulating the hydrolysis of GTP bound to its active form. We recently showed that IqgC plays an important role in the regulation of large-scale endocytosis. During cultivation in cell-culture dishes, we noticed that IqgC-null cells easily detach from the plastic surface, indicating that IqgC might be involved in the regulation of cell adhesion to the underlying substrata. We therefore proceeded to investigate the involvement of IqgC and its potential interactors in this process, using biochemical and microscopy approaches. Using the shaking assay, where cells were mechanically detached from the dish by rotational agitation, we confirmed that cells lacking IqgC are defective in the cell-substratum adhesion, an effect which could be rescued by expressing recombinant IqgC in IqgC-null cells. Cell tracking experiments showed that less adhesive IqgC-null cells migrate faster than wild-type. Somewhat surprisingly, reflection interference contrast microscopy showed slightly larger attachment area in IqgC-null than in wild-type cells. This effect might, however, be explained by our earlier finding that IqgC-null cells have a mild cytokinesis defect, leading to bigger, multinucleate cells. Total internal reflection fluorescence microscopy of cells expressing fluorescently labeled IqgC together with paxillin B or a probe for F-actin demonstrated that IqgC localizes to the paxillin B-containing adhesion foci. The pathways involved in the regulation of cell adhesion in D. discoideum are not yet fully elucidated, but it is hypothesized that the small GTPase Rap1 is one of the key regulatory proteins. Indeed, Rap1 was identified in the IqgC interactome as a potential IqgC binding partner and we confirmed a direct interaction between IqgC and Rap1 using yeast-two-hybrid, pull-down, and bimolecular fluorescence complementation assays. Here we show that IqgC protein is a positive regulator of the cell adhesion to the underlying substratum in Dictyostelium discoideum. Based on the presented results, we hypothesize that IqgC regulates adhesion via binding to small GTPase Rap1.

adhesion ; Dictyostelium ; IqgC

nije evidentirano