GRASP binding facilitates GTPase crosstalk in epithelial cell migration
The transition of epithelial cells from their normal non-motile state to a motile one requires the coordinated action of a number of small GTPases. Cell migration is stimulated by the coordinated activation of Arf and Rac GTPases. The crosstalk between these elements depends upon the assembly of a multi-protein complex that contains the Arf-activating protein cytohesin 2/ARNO and the Rac activating protein Dock180. The scaffolding protein GRASP binds directly both cytohesin 2 and Dock180 to coordinate their activities, and by doing so promotes crosstalk between Arf and Rac. These interactions were investigated using the Bi-Molecular Fluorescent complementation assay, based on the assembly of the N and C terminal fragments of Venus into a fluorescent protein. Neither VN nor VC is fluorescent by itself. YFP fluorescence is reconstituted if the two fusion partners interact, thereby bringing VN and VC into close proximity so that a functional YFP can be formed. Immunostaining of MDCK cells, showing the interaction of VN-cytohesin 2 and VC-GRASP as a positive control for the detection of YFP since GRASP and cytohesin 2/ARNO are known to interact via the coiled-coil domain of cytohesin 2/ARNO and the leucine rich region in GRASP. Cytohesin 2 is pseudo colored red, GRASP is pseudo colored blue, and the YFP fluorescence is shown in green.
Taken from: Lorraine Santy et al., 2013, BMC Cell Biology [View article]
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