YAP Regulation of RHO GTPase Activities Controls Epithelial Cell Morphology and Adhesion

Abstract

Yes-Associated Protein 1 (YAP) is the downstream effector of the Hippo pathway. Hippo is a kinase cascade that regulates development and tissue growth through phosphorylation of YAP and regulation of its localization. Nuclear YAP functions as a transcriptional coactivator with TEAD, regulating genes involved in tissue growth and homeostasis; when Hippo is active, YAP transcriptional activity is suppressed and YAP is localized to the cytoplasm. Previous studies from our lab have shown that loss of YAP perturbs cytokinesis in mammary epithelial cells, impairing membrane integrity and promoting genetic instability. In this thesis, we investigated the role of YAP in interphase epithelial cells. We observed that YAP knockdown in the MCF10A cell line promotes protrusive activity from acini in reconstituted basement membrane cultures. Similar protrusive behavior was observed by time-lapse microscopy in monolayer culture, where lamellipodia formation increased. Time-lapse microscopy also revealed that YAP knockdown impaired collective cell migration; cells dissociated and migrated individually instead of as a single sheet. This altered migratory behavior was associated with impaired junctional maturation. YAP knockdown affected the ratio of active RHO and RAC GTPase activities; RHOA activation was significantly decreased, and RAC activation was increased. Downstream of RHO, cellular contractility was also decreased in YAP knockdown cells. Interestingly, we found that expression of YAP mutants defective for transcriptional activity, such as TEAD-binding mutant YAP, or cytoplasmically-localized phosphomimetic YAP, was sufficient to restore wildtype acinar structure and cell contractility in cells depleted of YAP, suggesting that this function of YAP is independent of its transcriptional activity. Our findings suggest that cytoplasmic YAP plays an important role in regulating cell-cell adhesion and membrane protrusion when the Hippo pathway is active, and that alterations in cell behavior caused by translocation of YAP to the nucleus following Hippo inactivation can be mediated in part through loss of cytoplasmic YAP. This thesis highlights a role of YAP in maintenance of cellular morphology and reveals a novel function of YAP that is consistent with its role as a tumor suppressor.