The molecular mechanism of the core planar cell polarity complex elucidated with single-molecule imaging techniques in live Drosophila wing cells

The planar cell polarity (PCP) signaling pathway polarizes epithelial cells along an axis parallel to the epithelial sheet to generate front-back asymmetry at the tissue level. Failures in PCP signaling underlie developmental defects and diseases such as neural tube and heart defects, deafness, and contribute to cancer. Six core PCP proteins assemble into large, asymmetric complexes at cell-cell junctions, with Van Gogh and Prickle recruited to the proximal side of the cell-cell junction while Frizzled, Diego, and Dishevelled are recruited to the distal side. Flamingo forms asymmetric inter-cellular bridges connecting polarity between adjacent cell. Neither the potential requirement for clusters, nor their detailed organization are understood. To explore these questions, we used total internal reflection fluorescence microscopy to image GFP-tagged PCP proteins in the Drosophila pupal wing disc. Using this approach, we find that the molecular size distribution of PCP complexes follows an exponential function, suggestive of a single underlying growth mechanism. Mutations that block Dishevelled oligomerization decrease average cluster sizes, and also result in PCP phenotypic defects, demonstrating a requirement for large clusters in polarization. In conclusion, our findings provide a quantitative framework for understanding how cluster formation is coupled to the induction of tissue-level asymmetry during early embryonic development.