MOLECULAR ORGANIZATION OF MAMMALIAN EPITHELIAL TIGHT JUNCTIONS

Abstract

Conserved polarity protein complexes residing at the tight junctions (TJs) have been shown to mediate cellular polarity, signalling, and protein tracking. However, little is known about how these proteins are organized at or around the junctions. Here, we study the spatial segregation of the Par3 and Pals1 polarity proteins at mammalian TJs using the genetically-encoded APEX tag as a reporter for electron microscopy (EM). We show that Par3 localizes to the tight junctional plaque while Pals1 resides at the apicolateral border in a novel domain we call the vertebrate marginal zone (VMZ). We further performed APEX-mediated proximity ligation with subsequent SILAC-MS and show that we are able to spatially resolve the proximity proteomes of Par3 and Pals1, allowing us to put forward an interaction map based on this data. Finally, we uncovered a role of the VMZ in TJ formation and identified the Homer proteins as operating at the VMZ.