Desmosome dynamics: regulation via membrane rafts and dysregulation in pemphigus vulgaris Pubblico
Stahley, Sara Nicole (2015)
Abstract
Desmosomes provide adhesive strength to tissues such as the skin and heart by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Compromised desmosome function results in human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). In PV, autoantibodies (IgG) are directed against the desmosomal cadherin desmoglein 3 (Dsg3), resulting in the loss of desmosome-mediated cell-cell adhesion. We previously demonstrated that PV IgG induce membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft microdomains play a broader role by regulating the dynamics of both desmosome assembly and disassembly. In human keratinocytes, Dsg3 was found to be raft associated biochemically and colocalized with raft markers at cell borders by structured illumination microscopy (SIM), a form of super-resolution. Raft disruption prevented desmosome assembly and adhesion, indicating a functional link between rafts and desmosomes. Furthermore, PV IgG-induced desmosome disassembly occurred by a redistribution of Dsg3 into raft-containing endocytic linear arrays, resulting in cholesterol-dependent loss of adhesion. Thus, rafts are critical for both desmosome assembly and disassembly. Multiple PV pathomechanisms have been proposed based largely on in vitro studies. In order to better understand how PV IgG alters desmosome morphology and function in vivo, patient tissue was analyzed by SIM. Consistent with previous in vitro studies, we observed the following: aberrant clustering of desmosomal proteins, patient IgG colocalization with markers of rafts and endosomes, decreased Dsg3 levels and smaller desmosomes. Additionally, split desmosomes were detected in patient tissue, a finding not previously observed in cell culture models of PV. Desmosome splitting could be recapitulated in vitro by exposing cultured keratinocytes to both PV IgG and mechanical stress, demonstrating that desmosome splitting in patients is due to compromised desmosomal adhesion. These findings indicate that Dsg3 clustering and endocytosis are associated with reduced desmosome size and adhesion defects in PV patients. Defining the pathogenic alterations in PV patients provides a foundation for future in vitro studies investigating PV pathomechanisms and targeted therapeutics. Collectively, this dissertation demonstrates that raft membrane microdomains modulate the dynamics of desmosome assembly and disassembly, and that altered desmosome dynamics in PV result in the loss of desmosomal adhesion.
Table of Contents
Chapter 1: Dissertation overview...1
Chapter 2: Introduction...5
2.1 The epidermis...6
2.2 Desmosome ultrastructure and function...7
2.3 Molecular components and organization of the desmosome...9
2.3.1 Desmosomal cadherins...9
2.3.2 Plakoglobin...10
2.3.3 Plakophilins...11
2.3.4 Desmoplakin...12
2.3.5 Other desmosomal proteins...13
2.4 Tissue distribution of desmosomal proteins...14
2.5 Membrane rafts...15
2.5.1 Membrane raft definition...15
2.5.2 Membrane raft function...16
2.5.3 Studying membrane rafts...19
2.5.4 Rafts and cell junctions...21
Chapter 3: Compromised desmosomal adhesion in disease...30
3.1 Inherited disorders of the desmosome...31
3.2 Desmosomes in acquired disease...32
3.2.1 Pemphigus...32
3.2.2 Bullous impetigo / Staphylococcal scalded skin syndrome...37
3.2.3 Cancer...38
3.2.4 Other desmosomal diseases...40
3.3 Future directions...42
Chapter 4: Pathomechanisms of pemphigus: altered desmosome dynamics...47
4.1 Steric hindrance...48
4.2 Beyond steric hindrance...50
4.2.1 IgG targeting the Dsg EC3-5 domains play a role in acantholysis...50
4.2.2 PV IgG cause alterations in the desmosomal plaque...51
4.2.3 IgG-induced Dsg3 clustering...52
4.2.4 Dsg endocytosis and down-regulation...54
4.2.5 Cell signaling...55
4.2.6 Desmosome non-assembly vs. disassembly...58
4.3 Model for PV pathomechanisms and therapeutic implications...60
Chapter 5: Desmosome assembly and disassembly are membrane raft-dependent...65
5.1 Introduction...66
5.2 Results...69
5.2.1 Dsg3 and other desmosomal proteins are membrane raft associated...69
5.2.2 Desmosome assembly and adhesion are cholesterol dependent...70
5.2.3 Dsg3 raft partioning in associated with desmosome assembly...71
5.2.4 PV IgG-induced desmosome disassembly is raft-dependent...72
5.3 Discussion...73
5.4 Materials and methods...78
Chapter 6: Super-resolution microscopy reveals altered desmosomal protein organization in pemphigus vulgaris patients...95
6.1 Introduction...96
6.2 Results and discussion...99
6.2.1 PV IgG causes desmosomal protein clustering in patient tissue and in cultured keratinocytes...99
6.2.2 PV IgG associates with lipid raft-enriched linear array structures in vivo and is trafficked to endosomes...101
6.2.3 Desmosomes are smaller and split in PV patients...102
6.2.4 Mechanical stress causes desmosome splitting...103
6.2.5 Conclusions and implications...104
6.3 Materials and methods...105
Chapter 7: Summary and future directions...122
References...130
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