Macromolecular complexes are intricate structures that combine many different protein components to achieve a biological function. The relationship between complex architecture and function if often difficult to study because it requires bridging resolution scales between proteins, cells, and tissues. In the case of the cell-cell junction the desmosome, the complex is essential for adhering neighboring cells and maintaining tissue integrity in response to mechanical stress. Desmosomes can adopt two distinct adhesive states: calcium-dependent and calcium-independent adhesion, or hyperadhesion. Studying the relationship between desmosome organization and adhesive strength requires utilizing techniques with both molecular specificity and nanoscale resolution. To bridge this resolution gap, I utilized super-resolution microscopy to study the organization of the desmosome.
To investigate desmosomal plaque organization, I applied direct stochastic reconstruction microscopy (dSTORM) to determine the nanoscale structure of the desmosomal plaques. Desmosome architecture was altered in hyperadhesion induced by plakophilin-1 overexpression, which suggested a role for desmosome organization in determining adhesive strength. Another component of desmosome architecture is protein order, defined by a repeating array of proteins. I developed and applied fluorescence polarization microscopy (FPM) for measuring the order of the desmosomal cadherin desmoglein 3 (Dsg3) in living cells. While Dsg3 was ordered in calcium dependent desmosomes, inducing disassembly by removing exogenous calcium resulted in rapid disordering and suggested the importance of cadherin order for adhesion. Together these results suggested a role for the order and organization of desmosomes for controlling adhesive strength.
To further investigate this structure-function relationship, I measured the physical properties of desmosomes in both adhesive states. I induced hyperadhesion with a PKCα inhibitor, since the signaling mechanism for PKCα induced hyperadhesion has been previously characterized. Using dSTORM and FPM to measure order and organization in hyperadhesive desmosomes, I determined no difference in architecture between adhesive states. To explain the difference in functional states, I next investigated the role of protein dynamics. Desmosome proteins exhibited significantly decreased mobility in hyperadhesion, compared to calcium-dependent desmosomes. Ultimately, I concluded that loss of protein exchange is the mechanism of hyperadhesion. Together this dissertation highlights the roles of desmosome order, organization, and dynamics in controlling adhesive function.
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