All ETDs from UAB

Advisory Committee Chair

Alexa L Mattheyses

Advisory Committee Members

Susan L Bellis

Chenbei Chang

Rajeev S Samant

Joanne E Murphy-Ullrich

Document Type

Dissertation

Date of Award

2021

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Desmosomes are macromolecular junctions important in adhesion and resisting mechanical stress in epithelial and cardiac tissue. Desmosomes have a complex architecture with transmembrane cadherins, desmogleins and desmocollins, constituting the adhesive interface and plaque proteins, including plakoglobin and desmoplakin, binding the cadherin tails and integrating with intermediate filaments. Dysregulation of desmosomes occurs in many disease states, such as cardiomyopathies, skin diseases, and various cancers. While mature desmosomes' structure is generally understood, less is known about desmosome architecture during assembly and recycling. Desmosomes are dynamic, biochemically intractable, and diffraction-limited, making them challenging to study. To overcome these obstacles, I applied the super-resolution microscopy technique direct STocastic Optical Reconstruction Microscopy (dSTORM), which has approximately 20 nm resolution in the x-y plane. Using these super-resolution images, I quantified architectural changes to the proteins desmoplakin and plakoglobin during desmosome assembly. I used cell lines representing simple (MDCK), transitional (HUC), and stratified (NHEK) epithelia to determine universal features of desmosome architecture. To synchronize assembly, I utilized a switch from low to high calcium concentration, and then tracked desmosome 36 hours. I found a significant decrease in the distance between desmoplakin plaques but not plakoglobin plaques throughout maturation. This suggests a shift in how desmoplakin is arranged within desmosomes throughout maturation. The change in desmoplakin arrangement coincided with increased adhesive function and decreased E-cadherin enrichment. Finally, using two color dSTORM, I quantified the arrangement of desmoplakin in individual desmosomes characterized as E-cadherin positive or negative. Intriguingly, regardless of the time post-calcium switch, E-cadherin positive desmosomes had wider desmoplakin plaques. This indicates that nascent desmosomes, even those present at later time points, go through the same architectural changes. This ability to map protein localization throughout assembly at single desmosome resolution revealed a universal correlation of desmoplakin architecture with other maturation markers, suggesting the arrangement of desmoplakin may have functional implications. The novel approaches used here provide vital insight into the structure-function relationship in desmosome dynamics and have the potential to shed light on epithelial integrity in wound repair, development, and disease states.

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