P09. Cell shape and F-actin regulate Hippo signaling pathway

Abstract

The proliferation of cultured cells is regulated by cell density, a phenomenon known as cell contact inhibition of proliferation. Previously, we and others have shown that high cell density inactivates the transcription factor Tead, which controls cell proliferation, via the Hippo signaling pathway by suppressing nuclear localization of the coactivator protein Yap. Cell density affects both, cell–cell contacts and the cell shape, but the exact role of these latter cellular properties in regulating the Hippo pathway remains elusive. In this study, we examined the role of the cell shape on the Hippo pathway. To exclude the effects of cell–cell contacts, individual NIH3T3 cells were cultured in small cell–adhesive areas termed microdomains. The cell shape was changed according to the microdomains size. Nuclear localization of Yap was observed with large microdomains or corresponding flattened cells, but not with small microdomains resulting in the formation of round cells. Clear stress fiber formation was observed only in large microdomains concomitant with nuclear Yap. Disruption of F-actin by drug treatment reduced the nuclear localization of Yap and transcriptional activity of Tead irrespective of cell shape. Core components of the Hippo pathway (upstream to downstream) are Merlin, Mst, Lats, Yap and Tead. When activity of the Hippo pathway was blocked by the expression of a dominant negative form of Merlin, Yap stayed in the nucleus, even when F-actin was disrupted. These results suggest that F-actin regulates the Hippo pathway upstream of Merlin, and that the cell shape regulates the Hippo pathway independent of cell–cell contacts by controlling the amount of F-actin.