Desmosome Assembly and Disassembly: Lessons from Studying Dermatological Diseases Público
Caldara, Amber (Summer 2019)
Abstract
Cell-cell adhesion complexes mediate fundamental cellular interactions crucial in maintaining the integrity of skin, an essential protective barrier to the outside world. Desmosomes are large adhesion complexes critical for epidermal integrity and differentiation. These junctions are rigid enough to confer resistance to mechanical stress but dynamic enough to allow for processes such as wound healing and epidermal differentiation. Altered desmosomal adhesion results in the formation and progression of several genetic and acquired skin disorders. Additionally, desmosome stability is compromised during the process of epithelial to mesenchymal transition, a necessary step for tumors to metastasize. Thus, understanding the complex dynamics of desmosomal components and how desmosomes assemble and disassemble is essential for developing novel treatments for a variety of skin diseases.
Lipid rafts are sphingolipid and cholesterol rich membrane domains that introduce membrane heterogeneity and act as platforms for protein aggregation and signaling. All desmosomal proteins have been shown to associate with lipid rafts, however the biological significance of this association is unknown. We investigate the mechanism by which desmoglein (Dsg) associates with lipid rafts and uncover that the length of the transmembrane domain (TMD) is a key regulator of raft association. Additionally, we identify a patient with severe dermatitis multiple allergies and metabolic wasting syndrome that is caused by a point mutation within the TMD of Dsg1. This mutation abrogates lipid raft association of Dsg1, prevents Dsg1 incorporation into the desmosome, and results in Dsg1 retention in the Golgi apparatus. Additionally, we demonstrate that the bilayer within the desmosome is thicker than non-desmosome bilayer, thus identifying the desmosome as a lipid raft-like domain.
We then study the disassembly of desmosome through a pemphigus vulgaris model. Pemphigus vulgaris (PV) is an autoimmune disorder in which autoantibodies targeting Dsg3 and Dsg1 disrupt desmosome adhesion resulting in intraepidermal blisters. We determine that the sum of antibody titers against Dsg1 and Dsg3 is more correlative to disease severity than either titer on its own. We next demonstrate a relationship between Dsg3 autoantibody titers and an in vitro model to study the loss of cell-cell adhesion. These data demonstrate a relationship between Dsg antibody titer and disease severity and validate current methods for studying PV in cell culture. Together, these studies increase our understanding of how desmosomes assemble and disassemble, thus providing a platform for future investigation of the mechanisms regulating desmosome stability during tumorigenesis.
Table of Contents
Chapter 1: Focus and Impact 1
Chapter 2: The epidermis and cell-cell junctions. 5
2.1 The Epidermis. 5
2.2 Cell-Cell Complexes in the Epidermis. 7
2.2.1 Tight Junctions. 9
2.2.2 Gap Junctions. 10
2.2.3 Anchoring Junctions. 11
2.2.3.1 Adherens Junctions. 12
2.2.3.2 Hemidesmosomes. 14
2.2.3.3 Desmosomes. 15
2.3 The Desmosome. 15
2.3.1 Molecular components of the desmosome. 17
2.3.1.1 Desmogleins. 17
2.3.1.2 Desmocollin. 20
2.3.1.3 Desmoplakin. 21
2.3.1.4 Plakophilin. 22
2.3.1.5 Plakoglobin. 23
Chapter 3: Epithelial cell junctions and junctional proteins in tumorigenesis. 25
3.1 Epithelial to mesenchymal transition. 26
3.2 Tight junction proteins and cancer 28
3.3 Adherens junction proteins and cancer 28
3.4 Desmosome proteins and cancer 29
Chapter 4: Regulation of the desmosome. 33
4.1 Factors regulating the expression of desmosomal proteins. 33
4.2 Desmosome Stability. 35
4.3 Desmosome Assembly. 38
4.4 Desmosome regulation: Lipid rafts. 39
4.4.1 Lipid rafts. 39
4.4.1.1 Structure of Lipid rafts. 40
4.4.1.2 Function of lipid rafts. 42
4.4.3 Lipid rafts and desmosome function. 46
Chapter 5: The Desmosome is a Mesoscale Lipid Raft-Like Domain. 49
5.1 Abstract 50
5.2 Introduction. 50
5.3 Results. 53
5.3.1 Palmitoylation of Dsg3 is not required for lipid raft association. 53
5.3.2 The transmembrane domain of desmogleins mediates lipid raft association. 54
5.3.3 A mutation in the transmembrane domain of DSG1 causes severe dermatitis, multiple allergies, and metabolic wasting syndrome. 56
5.3.4 Disease causing mutation abrogates lipid raft association of Dsg1. 60
5.3.5 The lipid bilayer within desmosomes is thicker than non-desmosomal membranes 61
5.4 Discussion. 63
5.5 Materials and methods. 68
5.6 Acknowledgements. 77
Chapter 6: Pemphigus Vulgaris. 91
6.1 Pemphigus Overview.. 91
6.2 Clinic. 93
6.2.1 Etiology. 93
6.2.2 Diagnosis and Symptoms. 95
6.2.3 Measure of disease severity. 96
6.2.5 Enzyme Linked Immunosorbent Assays. 99
6.2.6 Treatment and Patient outcomes. 100
6.3 Bench. 103
6.3.1 Models and techniques to study PV.. 103
6.3.1.1 Human Tissue. 103
6.3.1.2 Mouse Models. 104
6.3.1.3 Keratinocyte Culture. 105
6.3.2 Mechanism of acanthylosis. 105
6.4 Outlook. 107
Chapter 7: Pemphigus Vulgaris Dsg ELISA scores are associated with PDAI and loss of keratinocyte adhesion in vitro. 108
7.1 Abstract 109
7.2 Introduction. 110
7.3 Results and discussion. 112
7.3.1 Epidemiology and clinical phenotype. 112
7.3.2 Total ELISA score correlates with PDAI. 113
7.3.3 DSG3 ELISA correlates with fragmentation assay. 114
7.3.4 PDAI correlates to disassociation assay for mucosal dominant patients. 116
7.3.5 Concluding remarks. 116
7.4 Materials and Methods. 117
7.6 Acknowledgements. 121
Chapter 8: Summary and future directions. 129
Section 8.1 Summary of dissertation work. 129
Section 8.2 Future directions. 130
8.2.1 Does Dsg3 endocytosis or loss of surface Dsg3 in keratinocytes after PV IgG treatment correlate to PDAI?. 130
8.2.2 What is the mechanism behind the thickening of the epidermis in SAM patients?. 132
8.2.3 How is DSG1(G578R) acting as a dominant negative?. 135
8.2.4 What is the role of lipid raft association in Dsg3 endocytosis?. 136
8.2.5 What are the mechanisms of Dsg3 clustering and linear array formation in Pemphigus Vulgaris? 138
Chapter 9: References. 148
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