The role of desmosome order, organization, and dynamics in adhesive function Público
Bartle, Emily (Spring 2019)
Published
Abstract
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.
Table of Contents
Chapter 1: Overview..................................................................................................................... 1
Chapter 2: Introduction............................................................................................................... 5
2.1 Desmosome function............................................................................................................. 6
2.1.1 Desmosome signaling.................................................................................................... 7
2.1.2 Desmosomes in disease.................................................................................................. 8
2.1.3 Calcium dependence of adhesive function................................................................... 12
2.2 Desmosome structure.......................................................................................................... 14
2.2.1 Cadherins...................................................................................................................... 14
2.2.2 Plaque proteins............................................................................................................. 17
2.2.3 Keratin.......................................................................................................................... 20
2.2.4 Complex organization.................................................................................................. 21
2.2.5 Desmosome organization by electron microscopy...................................................... 22
2.2.6 Desmosome organization by fluorescence microscopy............................................... 23
Chapter 3: Super-resolution and advanced imaging of epithelial junctions......................... 29
3.1 Introduction......................................................................................................................... 30
3.2 Structured Illumination Microscopy................................................................................... 33
3.3 Single Molecule Localization Microscopy......................................................................... 34
3.4 Stimulated Emission Depletion Microscopy....................................................................... 35
3.5 Fluorescence Polarization Microscopy............................................................................... 36
3.6 Fluorescence recovery after photobleaching....................................................................... 37
3.7 General Imaging Considerations for Super-resolution Microscopy................................... 39
3.8 Super-Resolution Microscopy of Epithelial Junctions........................................................ 41
3.8.1 Tight Junctions............................................................................................................. 41
3.8.2 Adherens Junctions...................................................................................................... 43
3.8.3 Gap Junctions............................................................................................................... 44
3.8.4 Desmosomes................................................................................................................. 46
3.8.5 Hemidesmosomes......................................................................................................... 48
3.9 Conclusion........................................................................................................................... 49
Chapter 4: Molecular organization of the desmosome by super-resolution microscopy..... 54
4.1 Introduction......................................................................................................................... 55
4.2 Results and Discussion........................................................................................................ 56
4.2.1 Desmosome plaques resolved by dSTORM................................................................. 56
4.2.2 Molecular map of the desmosome by dSTORM.......................................................... 57
4.2.3 Reorganization of plaque proteins in PKP-1-mediated hyperadhesive desmosomes.. 60
4.2.4 Desmosome molecular organization is conserved in skin........................................... 61
4.3 Materials and Methods........................................................................................................ 62
Chapter 5: Desmoglein 3 order and dynamics in desmosomes determined by fluorescence polarization microscopy................................................................................................................................... 79
5.1 Introduction......................................................................................................................... 80
5.2 Results................................................................................................................................. 83
5.2.1 Theory of protein order in desmosomes....................................................................... 84
5.2.2 Desmoglein 3 extracellular domain is ordered............................................................. 86
5.2.3 Dsg3 order decreases prior to loss of cell adhesion..................................................... 88
5.3 Discussion........................................................................................................................... 92
5.4 Materials and Methods........................................................................................................ 95
Supplemental Figure 9. Calcium switch time course of Dsg3-link-GFP................................ 114
Chapter 6: Protein exchange regulates calcium independence of epithelial junctions...... 116
6.1 Introduction....................................................................................................................... 117
6.2 Results............................................................................................................................... 120
6.2.1 Dsg3 order is not required for adhesive function....................................................... 120
6.2.2 Desmosome architecture is unaltered in hyperadhesion............................................ 123
6.2.3 Desmosomal cadherins are stabilized by hyperadhesion........................................... 124
6.2.4 Plaque proteins are stabilized in hyperadhesion........................................................ 126
6.2.5 Trans-binding is necessary for Dsg3 order and loss of mobility............................... 127
6.2.6 Phosphorylation of DP S2849 is the molecular switch controlling desmosome protein stability in hyperadhesion...................................................................................................................... 129
6.3 Discussion......................................................................................................................... 131
6.4 Materials and Methods...................................................................................................... 135
Chapter 7: Summary and future directions........................................................................... 163
7.1 How does the nanoscale organization of the desmosome contribute to adhesive function? 165
7.2 What is the role of cadherin order in desmosome adhesion?........................................ 167
7.3 How can the physical properties of desmosomes inform the study of cell functions?. 169
7.4 Concluding remarks...................................................................................................... 171
References................................................................................................................................... 173
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