Ultrastructure of adherens junctions [completed project]

Despite three decades of intense research on cadherin biology we know shockingly little about the physical structure of adherens junction. How are the cadherins arranged within the membrane? how are they connected to the cytoskeleton? Are the adaptors and regulators organized in any particular way? The reason for our ignorance has to do with the limitation of our observation methods: there is a gap in our ability to visualize objects between the resolution of X-ray diffraction crystallography (~0.1nm) and the resolution of conventional light microscopes (~300nm). As it happens, the scale of protein complexes, which is what the adherens junction is made of, is in the size range of 30-100 nm – right smack in our blind spot! Electron microscopy has adequate resolution, but sample preparation of adherens junctions is a challenge and its difficult to label proteins so it is difficult to know what you are looking at in the electron densities.

Thankfully, in the past couple of years, a new method of microscopy was invented, which cleverly overcomes the diffraction limit of light and enables imaging immunolabeled cells down to a resolution of 10-30nm. This method, referred to as super resolution microscopy, depends on taking multiple (up to 20,000) separate images of the same sample, each time recording only a small subset of fluorescent proteins or antibody-dyes. When only a single molecule is emitting light it is possible to pinpoint its location at very high resolution. After processing of all the images, a composite image that contains all the light sources (proteins) at high resolution is presented.

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In close collaboration with Tony Kanchanawong, we used 3D-STORM and iPALM to decipher the ultrastructure of cadherins, actin and a number of important adaptor and signaling proteins at adherens junction in various stages of assembly and under varying conditions of tension. We are also testing the effect of different mutations affecting cadherin binding on the organization of cadherins and ultrastructure of the junction. We found that loosely organized clusters of approximately five E-cadherin molecules that form independently of cis or trans interactions, and which are delimited by the cortical F-actin meshwork, are the precursors of trans-ligated adhesive clusters that make up the adherens junction.

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This work was published in Developmental Cell 32(2):139-54. Yao2015compressed See also MBInsights: Defining adhesion clusters

We also collaborated with Pakorn (Tony) Kanchanawong‘s group on elucidating the 3D stratification of adherens junctions formed between MDCK or C2C12 cells and a cadherin-coated surface using super resolution microscopy. This work revealed a separation between the membrane proximal cadherin-catenin layer and an actin-rich compartment that is bridged by an interface zone containing vinculin. The study also elucidated the mechanism of vinculin activation at adherens junctions, which entails both mechanical tension AND tyrosine phosphorylation.

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This study was published in Nature Cell Biology doi: 10.1038/ncb3456.