Celsr1-mediated planar cell polarity: defining the adhesive interface and mechanisms of asymmetry

Project Summary Planar cell polarity (PCP) is a core cell contact and signaling pathway that is largely conserved from Drosophila to mammals, and is fundamental to early development and tissue organization in complex, multicellular organisms. The long-term goal of this study is to further our understanding of how central PCP protein, Celsr1, mediates adhesive interactions required for the establishment and maintenance of PCP. Acting orthogonally to apico-basal polarity, PCP is the collective and organized polarization of cells along a tissue plane. A striking example of PCP is the ordered alignment of body hairs on mammalian skin. Core components of this pathway include: Celsr (Flamingo/Fmi), Frizzled (Fz), Vangl (VanGogh/Vang), Dishevelled (Dvl), Prickle (Pk) and Diego (Dgo). A hallmark feature of PCP is the asymmetric localization of Fzd/Dv/Dgo and Vangl/Pk at cell borders within a junctional complex organized via intercellular interactions of Celsr, a large atypical cadherin and member of the GPCR super-family. Disrupting Celsr function in mice leads to a range of detrimental phenotypes including neural tube defects, abnormal hair patterning and embryonic lethality. Further, multiple Celsr mutations in humans have been associated with neural tube defects and diseases such as spina bifida and cardiomyopathies. While important developmental roles for Celsr at the tissue-level have been established with mouse and fly studies, an understanding of Celsr function at the molecular and cell level is lacking. To elucidate how PCP asymmetry is achieved, we need to define the mechanisms of Celsr1 extracellular adhesion. The mouse skin is an ideal model system to investigate the conserved mechanisms of PCP and our lab has recently established a spontaneously polarizing in vitro, organotypic model system for assaying PCP asymmetry and function. Super-resolution imaging techniques such as structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) further add to our robust toolbox to study epidermal PCP. The overall goal of this proposal is to investigate how the principle PCP protein, Celsr1, mediates extracellular adhesion to coordinate functional PCP. We hypothesize that Celsr1 cadherin repeats mediate intercellular and lateral adhesive interactions required for asymmetrical PCP protein localization and function. Understanding how individual cell-cell interactions at the molecular level contribute to tissue structure and function will fill a substantial and fundamental knowledge gap in the PCP field. This proposal integrates molecular biology, biochemistry and advanced microscopy approaches to elucidate mechanisms of PCP asymmetry and function in vitro and in vivo. Completion of these studies will provide novel insight into the mechanisms the regulate PCP in the mammalian epidermis at the most fundamental level and illuminate our conceptual understanding of PCP complex formation and function.