Molecular mechanism of core planar cell polarity complex function elucidated with single-molecule methods.

Abstract

The planar cell polarity (PCP) signaling pathway polarizes epithelial cells along an axis parallel to the epithelial sheet in order 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 Vang and Prickle recruited to one side of the cell-cell junction while Frizzled and Dishevelled are recruited to the other. How this asymmetric assembly process occurs is not known. Genetic evidence suggests that the process of symmetry breaking at PCP clusters is coupled to cluster formation. To test how this occurs, we developed a method to count the number of each of the six core proteins in individual PCP complexes. Specifically, by analyzing bleaching traces obtained with a high frame rate in live Drosophila wing cells imaged using total internal reflection microscopy, we quantify the intensity of single fluorophores in each complex and thereby calculate the molecular number in the corresponding PCP complex. Using this approach, we find that the molecular size distribution of PCP complexes follows an exponential function, indicating a single underlying growth mechanism. We report that specifically, the number of Vang molecules in PCP complexes increases from 15 hours after puparium formation until reaching a steady state around 28 hours. Besides, we measure the asymmetry as a function of cluster size and test the dependence of the observed relationship by introducing gain and loss of function mutations that perturb cluster assembly. In conclusion, our findings provide a detailed understanding of how the PCP mechanism generates stable polarization during embryonic development.