Cell Polarity Scratch and See

John F. Foley

Science’s STKE, AAAS, Washington, DC 20005, USA

Scratching monolayers of tissue culture cells and observing the changes that occur in those cells closest to the scratch have provided important insights into how cells polarize and coordinate their movement to repair the damage. The area of the cell closest to the scratch (essentially the new front of the cell) becomes the site of actin-rich protrusions, whereas rearrangements of the microtubule cytoskeleton push the centrosome and Golgi toward the front of the cell and push the nucleus toward the back. This polarization of the cell is dependent on the activity of the Rho family guanosine triphosphatase, Cdc42, which also leads to the assembly of a complex between the scaffold protein Par6 and atypical protein kinase C (aPKC). This complex is necessary for the accumulation of adenomatous polyposis coli (APC), a tumor suppressor protein involved in Wnt signaling. Inhibition of the activity of glycogen synthase kinase (GSK)-3 by PKC phosphorylation is presumed to allow the accumulation of APC at the leading edge. However, experiments by Schlessinger et al. suggest that the interaction between GSK-3 and aPKC is not the relevant one. They generated double knockin mice (GSK-3^SA) in which the relevant Ser residues of both GSK-3 and GSK-3 were mutated to Ala residues, thus preventing their inhibition by phosphorylation. Unexpectedly, movement of the Golgi and centrosome in response to monolayer scratching of fibroblasts from GSK-3^SA mice was normal. Dishevelled (Dvl) is another component in the Wnt pathway, and Wnt stimulates the binding of Dvl to the scaffold protein axin, which results in the release of GSK-3 from a complex containing axin, APC, and -catenin. Knockdown of Dvl or axin with siRNA in rat embryo fibroblasts inhibited reorientation of both the centrosome and Golgi, as did treatment of scratched monolayers with a Wnt-binding protein, demonstrating the importance of Wnt signaling in cell polarization. Inhibition of Wnt5a, but not Wnt1 or Wnt3a, by siRNA blocked reorientation of the fibroblasts in scratched monolayers and also inhibited the accumulation of APC to microtubules at the leading edge, as demonstrated by confocal microscopy. Western blotting demonstrated that scratching of the monolayer resulted in the phosphorylation of Dvl proteins that was dependent on Wnt signaling. Finally, coimmunoprecipitation studies showed that aPKC and Dvl2 formed a complex after scratching of monolayers, which was dependent on both Wnt and Cdc42, as demonstrated by the use of Wnt-specific siRNA and an inhibitor of Cdc42. Thus, Cdc42-dependent Wnt signaling appears to play a role in cell polarization.

K. Schlessinger, E. J. McManus, A. Hall, Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity. J. Cell Biol. 178, 355-361 (2007). [Abstract] [Full Text]

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