Epithelial (E)-cadherin receptors form adhesive bridges linking cells into tissues, and through interactions of their cytoplasmic tails with the actin cytoskeleton they provide mechanical coupling between neighbouring cells, which is essential for epithelial morphogenesis and homeostasis. Crystal structures of E-cadherin extracellular domains exhibit a lattice with specific cis and trans interactions. However, the density of E-cadherin measured at adherens junctions is far below the crystal lattice packing. Addressing this conundrum has been difficult because the structure of adherens junctions is not resolvable by diffraction-limited microscopy. Here, we used superresolution microscopy to elucidate the nanoscale architecture of adherens junctions in mammalian epithelial cells at ∼30nm resolution, and evaluated the contributions of the cytoplasmic tail and filamentous (F)-actin as well as extracellular interactions. We found that both apical and lateral junctions are composed of discrete clusters, the majority of them composed of 3-10 Ecadherin receptors, with densities about 10-fold lower than the crystal packing density. Surprisingly, such clusters could form independently of trans- or cisinteractions, but the majority of apical clusters were adhesive and contained sub-domains with densities befitting crystal lattice packing. In agreement with this observation, differential labeling of E-cadherin in neighbouring cells revealed that while the proportion of adhesive clusters in apical junctions was high, only a subset of lateral clusters were adhesive. Importantly, we could detect densities consistent with the crystal structure lattice at the core of adhesive clusters, which were dependent on extracellular domain interactions. Strikingly, we discovered that the cytoplasmic tail of E-cadherin limited cluster size, and determined this to be a result of its interaction with F-actin. Dual-colour super resolution imaging revealed a mutually exclusive localization, in which E-cadherin clusters were surrounded by an F-actin meshwork. Depolymerizing F-actin, deletion of cytoplasmic tail of E-cadherin, as well as strengthening the trans-interaction led to a growth in E-cadherin cluster size. Furthermore, formin inhibition disrupted actin structures at AJs as well as cortex, leading to enlarged cell area, but shortened cell height and squeezed junction size. And the F-actin stripes came closer to the cytoplasmic tails of E-cadherin after formin inhibition. These findings suggest that the basic unit of E-cadherin adhesion is a cluster composed of a small number of E-cadherin receptors, which are loosely packed within each cluster and corralled by the cortical F-actin meshwork. Such precursors then become trans-ligated and forms mature apical junctions. Thus, our results elucidate the nanoscale architecture of adherens junctions, as well as the molecular mechanisms driving its assembly.
|School:||National University of Singapore (Singapore)|
|School Location:||Republic of Singapore|
|Source:||DAI-B 77/06(E), Dissertation Abstracts International|
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