Characterizing Vangl2's Interaction with Celsr1 And Investigating the Function of Neural Tube Defect-Associated Vangl2 Mutants

Abstract

Despite advancements in prenatal care, neural tube defects (NTDs) continue to be one of the most prominent and fatal disorders affecting fetuses. While dietary insufficiencies have been shown to affect proper neural tube closure, it is likely that there is a genetic, heritable trait that impacts disordered neurulation. The planar cell polarity (PCP) pathway plays a fundamental role in ordering cells across a tissue, and mutations in PCP components have been implicated in cases of human NTDs. One such protein is Vangl2, which associates with the atypical cadherin Celsr1. In the presence of Celsr1, Vangl2 asymmetrically localizes to lateral intercellular junctions in vivo, and in cultured mouse keratinocytes, Vangl2 enriches at cell-cell junctions. Loss of any of the core PCP proteins leads to defective PCP signaling and has been shown to cause developmental defects. Although PCP is instrumental in many embryological processes, the nature of the association between Vangl2 and Celsr1 in vertebrates is still largely unknown. There also exists a substantial gap in knowledge on the functional changes that occur when Vangl2 is mutated in mammalian systems. This study employed chimeric Vangl2 and Celsr1 proteins, with wild-type protein domains replaced with those of proteins not involved in PCP, to investigate which domain(s) of each protein were required for the proper recruitment of Vangl2 to intercellular junctions in cultured keratinocytes. Additionally, phosphorylation mutations and mutations derived from human NTD-defect cases were introduced into Vangl2, and the junctional recruitment levels of each were tested. This study shows that Celsr1’s C-terminal tail is necessary and sufficient for wild-type Celsr1 levels of Vangl2 recruitment and demonstrates that, while each of the protein domains of Vangl2 is required for wild-type level enrichment, the loss of Vangl2’s C-terminal tail abrogates recruitment. Additionally, the data from the experiments conducted on Vangl2 point mutants show that the loss of the phosphorylatable residues S11 and Y308 reduce Vangl2 recruitment, while the NTD-derived Vangl2 mutation F437S completely inhibits Vangl2 junctional enrichment. These findings clarify the physical interaction domains of Celsr1 and Vangl2 and demonstrate the functional effects of Vangl2 mutations identified in previous studies.