Regulation of Apical Polarity Complexes.

Abstract

Epithelial tissue lines exterior surfaces of the body and organ compartments, providing a multitude of functions including filtration, barrier establishment, and secretion. Epithelial cells are arranged in well-organized sheets and display a distinct architecture. Notably, epithelial cells are polarized: their plasma membranes are segregated into distinct apical and basolateral membrane domains by tight junctions. This membrane polarization is essential for proper epithelial function, and loss of polarization leads to developmental defects, cancer, and fibrotic disease. Apical polarity complexes control epithelial polarization by promoting tight junction formation. Our research focus is the tripartite apical polarity complex consisting of Crumbs3a (Crb3a), Protein associated with Lin-7-1 (PALS1), and PALS1 associated tight junction protein (PATJ). Little is known about the regulation of Crumbs complex proteins and how this regulation contributes to epithelial polarization. Here, we investigated the regulation of the Crumbs complex in the establishment and maintenance of epithelial polarity using molecular techniques and biochemical assays in cell culture models of epithelia. First, we discovered that reducing cellular levels of Lin-7 (a PALS1 binding partner) results in tight junction defects including delays in junction formation, mislocalization of tight junction proteins, and increased degradation of PALS1 and PATJ. These results suggest that Lin-7 regulates the Crb3a-PALS1-PATJ module by stabilizing components of this complex. Next, we found that Crb3a message and protein are highly regulated. Specifically, Crb3a transcription is repressed by the transcription factor Slug in depolarized thyrocytes. In addition, we showed that Crb3a is essential for establishing the apical membrane via trafficking through Rab11a positive endosomes in kidney epithelial cells grown in three-dimensional culture. Interestingly, once apical membranes are established, Crb3a bypasses the apical recycling system and may enter the multi-vesicular body pathway. Also, we demonstrated that the half-life of Crb3a is not stabilized by the presence of an apical surface. Furthermore, we find Crb3a protein exists in both highly mobile membrane and tight junction populations suggesting that polarization itself is a dynamic process. Taken together, our data suggest that the Crumbs polarity complex is subject to multiple levels of regulation in the cell in order to carefully control cellular growth, morphology, and function.