Small GTPase Regulation of Epithelial Cell Motility
Epithelial cells are normally stationary and non-motile, however they become migratory during wound healing, tissue morphogenesis, and metastasis of epithelial tumors. Cellular migration requires precise spatial and temporal coordination of alterations in cellular signaling and architecture. The long-term goal of the Santy lab is to understand the signals and processes leading to the initiation of motility in epithelia cells, focusing particularly on small GTPase cascades.
Small GTPases, particularly members of the Rho family, are well-established regulators of cell migration. Recently another small GTPase, ARF6, has emerged as key regulator of cell movement. ARF6 regulates the endocytosis and recycling of some plasma membrane proteins. It also initiates remodeling of the cortical actin cytoskeleton, and is activated in response to a number of pro-migratory growth factors.
GTPases are turned on by guanine nucleotide exchange factors (GEFs). We have shown that one ARF-GEF, ARNO/cytohesin-2, plays a central role in the regulation of epithelial motility. Our current work is focused on defining the functions of cytohesin-2 that underlie its ability to initiate movement in epithelial cells.
Cytohesin-2/ARNO and ARF6 signaling pathways that regulate epithelial motility
Overexpression of cytohesin-2/ARNO leads to a robust enhancement of epithelial migration. This enhanced motility requires the activation of ARF6 by cytohesin-2. Subsequently ARF6 activates Rac and phospolipase D, which are both independently required for enhanced motility. We have shown that ARF6 acts via the Rac-GEF Dock180 to turn on Rac. Recently, we have found that two scaffold proteins, GRASP and IPCEF1, coordinate to assemble a larger protein complex that contains both cytohesin-2 and Dock180. Rac activation requires the assembly of this complex and activation of ARF6. Rac activation promotes cytoskeletal remodeling and cell shape change. Current work in the lab seeks to further define this cytohesin-2/Dock180 complex and to understand how its assembly is regulated.
Other projects in the lab are focused on identifying the signal transduction pathways that are activating cytohesin-2/ARNO and ARF6 to promote epithelial movement. We are particularly interested in signaling in response to hepatocyte growth factor (HGF). HGF induces movement in a large number of epithelial cell lines. Treatment with HGF produces scattering of cells on a flat surface, and branching morphogenesis in 3D culture. We are working to define the pathway leading from HGF to ARF6 and to understand how this signaling is spatially controlled.
Cytohesin-2/ARNO and ARF6 regulated trafficking in epithelial motility
In addition to remodeling the cortical actin cytoskeleton and changing cell shape, ARF6 also regulates the endocytosis and recycling of adhesion receptors such as β1 integrin. The appropriate arrangement of adhesions converts contraction into movement. Recently, we have determined that cytohesin-2 but not the closely related protein, cytohesin-3, is required for β1 integrin recycling, adhesion and migration. This is the first demonstration of a unique action by one of these cytohesins. We are currently working to define the critical differences between cytohesin-2 and cytohesin-3. We are also interested in determining the precise compartment where cytohesin-2 acts to promote β integrin recycling.
Figure 1. The coiled-coil domain of ARNO is necessary for the induction of epithelial motility. A) MDCK cells were infected with adenoviruses encoding the indicated ARNO constructs for three hours. Cells were then fixed and stained with mouse anti-myc followed by Alexa-488 conjugated anti-mouse antibody and rhodamine-phalloidin. Control cells were infected with adenovirus encoding wild-type ARNO in the presence of doxycycline to suppress transgene expression. Bar equals 50 µm. B) Motility of cells expressing the indicated constructs was tested using the transwell assay. The percent of cells migrating through the filter in 18 hours are indicated. Data shown are mean ± standard deviation of triplicate samples. C) Expression levels of the myc-tagged ARNO constructs and actin in the cells subjected to the transwell assay shown in B, were visualized by Western blot of saved cell samples with mouse anti-myc and mouse anti-actin antibodies.
Figure 2. Integrin β1 recycling requires Cytohesin-2/ARNO but not Cytohesin-3/GRP1. (A) Cell surface β1 integrin was labeled with antibody, internalized and recycling initiated in MCF-7 cells transfected with siRNA targeting ARNO or GRP1. Retained internal β1 integrin was isolated and visualized by Western blot. (B) The percent of internal integrin β1 5 min after stimulation was determined by comparison to a 0 min stimulation sample. Data shown are mean ± standard error of >3 separate experiments. Levels of internal integrin β1 at 5 minutes in the ARNO or GRP1 knockdown cells were compared to the levels in control cells using a T-test. Double asterisk indicates P<0.01. (C) Surface and Total integrin β1 was visualized in MCF-7 cells transfected with siRNA targeting ARNO or GRP1 after various times of recycling. Bar, 25µm