The Transforming Rho Family GTPase Wrch-1 Disrupts Epithelial Cell Tight Junctions and Epithelial Morphogenesis

Abstract

Wrch-1, an atypical and transforming Rho GTPase, regulates cellular activities including proliferation and actin organization, but its functions and effectors remain poorly characterized. We show here that Wrch-1 distributes along the apical and basolateral membranes in MDCK cells and binds the cell polarity protein Par6 in a GTP-dependent manner. Activated Wrch-1 negatively regulates the kinetics of tight junction (TJ) assembly during epithelial cell polarization but has no detectable effect on overall cell polarity in confluent monolayers. It also causes a dramatic cytoskeletal reorganization and multilayering in cells grown in two-dimensional culture and disrupts cystogenesis of cells grown in three-dimensional (3D) culture. Similarly, short hairpin RNA-mediated knockdown of Wrch-1 perturbs cystogenesis in 3D culture, suggesting that tight regulation of Wrch-1 activity is necessary for normal epithelial morphogenesis. A weakly transforming effector domain mutant of activated Wrch-1 that inhibits Par6 binding abrogates the ability of Wrch-1 to disrupt TJ formation, actin organization, and epithelial morphogenesis. We hypothesize that Wrch-1-induced morphological and growth transformation may occur in part through Par6-mediated disruption of TJs and actin organization.

ACKNOWLEDGMENTS

We thank Victoria Bautch (UNC-CH) for antibodies, Christopher Johnston (UNC-CH) for assistance with protein purification, and Sam Wolff (UNC-CH) for guidance in IF and TER experiments.

This work was supported by National Institutes of Health grants to A.D.C. (CA109550) and to A.S.F. and James M. Anderson (DK61397). J.K.A. was supported by NIH training grant T32-GM008581, and D.C.B. was supported by NIH training grants T32-GM007040 and T32-CA071341.