Biophysics of cadherin interactions and junction assembly

Abstract

<p>Desmosomes are robust cell adhesion junctions that are composed of two transmembrane desmosomal cadherin proteins: desmoglein (Dsg) and desmocollin (Dsc). The extracellular regions of Dsc and Dsg form an adhesive interface between cells while their cytoplasmic tails links to the intracellular keratin filament network. This dissertation investigates how Dsc and Dsg assemble into desmosomes using single molecule force measurements with an Atomic Force Microscope (AFM) and cell-based fluorescence assays. In Chapter 1, I give a brief over view of the content of this dissertation and the principles of AFM force measurements. In Chapter 2, I characterize the binding of isoform 2 of desmosomal cadherins. I show that Dsc2 dimerizes homophilically in a Ca2+ and tryptophan-2 (W2) dependent fashion; this binding mechanism, called ‘strand-swap dimerization’ has previously been found with other cadherins. In contrast, Dsg2 forms Ca2+ and W2 independent heterophilic binding with Dsc2. In Chapter 3 of the thesis, I describe how Dsg2 is recruited to desmosome. I show that E-cadherin (Ecad), a classical cadherin, interacts with Dsg2 in a Ca2+ independent manner, via a conserved Leu 175 on the Ecad cis binding interface. Furthermore, we demonstrate that desmosome assembly is initiated at sites of Ecad trans homodimerization and that Ecad-L175 is required for efficient Dsg2 and desmoplakin (DP) recruitment. Our data suggest that Ecad trans interactions at nascent cell-cell contacts initiate the recruitment of Dsg2 through direct cis interactions with Ecad; consequently, Dsg2 binds to Dsc2 and mediate robust desmosome assembly. In the fourth Chapter of this thesis, I develop and use a fast fluorescence recovery after photobleaching (FRAP) method to identify the dynamics of the Ecads trafficking at cell-cell junctions. Our preliminary data suggests that Ecads are recycled in and out of the junction by vesicle fusion. The final Chapter summarizes the conclusions and I propose future directions for these projects.</p>