Understanding The Regulatory Mechanisms Underlying the Establishment of Mammalian Planar Cell Polarity Through Biochemical and Cell-Based Approaches

Abstract

Planar Cell Polarity (PCP) refers to the polarization of cells within a tissue plane that arises from the asymmetric partitioning and clustering of the core PCP proteins to opposing cell boundaries. It is a critical phenomenon that drives the morphogenesis of vital mammalian organs. PCP arises in cells within epithelial tissue, such as the epidermis, when the transmembrane proteins Celsr1, Frizzled (Fz) and the cytoplasmic protein, Dishevelled (Dvl), partition to the posterior side of the cell boundary, and the transmembrane proteins Celsr1, Van Gogh (Vang), and the cytoplasmic protein, Prickle (Pk), partition to the anterior side of the cell boundary, starting from an initially uniform distribution. While PCP has been extensively studied using genetic models to perturb the function of the individual proteins, a mechanistic understanding of the protein-protein interactions that drive PCP protein partitioning, and the regulatory mechanisms underlying such behavior, is lacking. In vitro biochemical characterizations of these protein-protein interactions and reconstitution of PCP are, therefore, powerful approaches towards understanding the biochemical mechanisms underlying PCP protein partitioning. To that end, purification of a truncated Dvl3 construct and a full-length Pk1 construct was optimized and successfully accomplished. Furthermore, the PCP proteins are known to be heavily phosphorylated, with Casein Kinase 1-mediated phosphorylation of these proteins implicated in PCP establishment in polarized tissues. Therefore, a realistic recapitulation of PCP protein partitioning in vitro will rely on a comprehensive understanding of the phosphorylation of these proteins and its effects on their asymmetric partitioning and clustering at junctions. To that end, extensive analysis of the effects of Casein kinase 1ε/δ-inhibition of these proteins on their enrichment at junctions revealed a sensitivity of the junctional stoichiometry of these proteins on their Casein kinase 1ε/δ-mediated phosphorylation – a parameter that is critical for proper PCP establishment.