Mechanism of Transmembrane Domain Driven Desmoglein Raft Association in Desmosome Assembly Open Access

Zimmer, Stephanie (Summer 2021)

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Desmosomes link cytoskeletal elements of adjacent cells through a series of protein-protein interactions to confer robust mechanical cell adhesion and promote epidermal integrity. Mutations in the genes encoding the desmosomal proteins, such as desmoglein-1 (DSG1), can cause skin fragility diseases such as palmoplantar keratoderma (PPK) or severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. The desmosome has been characterized as a mesoscale lipid raft membrane microdomain whose assembly relies on raft association. Lipid rafts are sphingolipid- and cholesterol-enriched functional ordered plasma membrane regions, yet the relevance of the desmosome as a lipid raft and the mechanism driving raft association of the desmosomal proteins remains unclear. Physical properties of single-pass transmembrane domains (TMDs), including length, exposed surface area, and palmitoylation drive raft association. Furthermore, we have identified disease-causing DSG1TMD mutations in patients with PPK and SAM syndrome which obstruct these properties. Therefore, we hypothesize that DSG1TMD physical properties dictate raft association and desmosome assembly while mutations disrupting these properties cause disease. We designed and expressed a panel of Dsg1TMD-GFP variants in DSG-null cells to individually assess the contribution of each physical property towards Dsg1 raft association, desmosome assembly, and function. TMD length and exposed surface area but not palmitoylation contributed to DSG raft association which correlated with desmosome quantity and function. However, we could not rule out whether a motif within the Dsg1TMD sequence contributed to raft association. Furthermore, we identified a Dsg1TMD-GFP variant with a scrambled sequence which uncoupled raft association from desmosome assembly and function. We propose an amended model of desmosome assembly in which raft association of desmosomal cadherins in concert with protein-protein interactions drives segregation from adherens junctions while promoting further desmosome assembly by stabilizing nascent desmosomal clusters. We start with a detailed overview of the current understanding of desmosome assembly followed by a description of our DSG-null model and the use of this model to differentiate pathomechanisms of two disease-causing DSG1TMD mutations. We characterize the Dsg1TMD variant panel and then conclude with a series of future directions to address the new questions uncovered by this work.

Table of Contents

Chapter 1

The Desmosome as a Model for Lipid Raft Driven Membrane Domain Organization 1

1.1    Plasma membrane organization and intercellular junctions 2

1.2    Skin and heart require desmosomes to resist mechanical stress 5

1.3    The desmosome has features characteristic of a lipid raft-like membrane domain 8

1.4    Mechanism of desmosomal protein association with lipid raft membrane microdomains 10

1.4.1    TMD Length 10

1.4.2    TMD Surface Area 11

1.4.3    Palmitoylation 12

1.5    Role of raft association in disease 14

1.6    A new model for epithelial intercellular junction organization: lipid rafts as a driving force for the assembly and segregation of junctional complexes 15

1.7    Acknowledgements 19

Chapter 2

Establishing a New Model System to Study Desmoglein Function 20

2.1 Introduction 21

2.2 Results 22

2.2.1 DSG-null cell characterization reveals desmosomal defects 22

2.2.2 Exogenous expression of Dsg species rescues desmosomal defects in DSG-null cells 25

2.3 Discussion 27

2.4 Materials and Methods 28

2.5 Acknowledgements 31

Chapter 3

Differential pathomechanisms of DSG1 Transmembrane Domain Mutations in Skin Disease 32

3.1 Introduction 34

3.2 Results 36

3.2.1 Dsg1TMD mutants support the formation of fewer, smaller, and weaker 36

3.2.2 Dsg1TMD mutants reduce desmosome assembly by disrupting raft association 38

3.2.3 Dsg1PPK and Dsg1SAM exhibit distinct trafficking defects 41

3.2.4 Low Dsg1PPK expression levels are caused by increased protein turnover rates 43

3.3 Discussion 44

3.4 Materials and Methods 48

3.5 Acknowledgements 53

Chapter 4

The Desmoglein-1 Transmembrane Domain Drives Raft Association during Desmosome Assembly 55

4.1 Introduction 56

4.2 Results 57

4.2.1 Dsg1TMD variants are predicted to maintain α-helical structures characteristic of TMDs 57

4.2.2 Dsg1TMD-GFP variants mostly localize to cell borders 60

4.2.3 A full-length TMD is required for Dsg1 raft association, desmosome assembly, and desmosome function 64

4.2.4 TMD palmitoylation does not contribute to Dsg1 raft association or desmosome assembly 68

4.2.5 Altering Dsg1TMD surface area reduces raft association, desmosome assembly, and function 71

4.2.6 Scrambling the Dsg1 TMD sequence reduces raft association but has differential effects on desmosome assembly and function 75

4.2.7 Dsg1 raft association predicts desmosome function 79

4.3 Discussion 81

4.3.1 Dsg1 TMD properties and raft association 83 TMD Length 84 Palmitoylation 86 Exposed Surface Area 86

4.3.2 Dsg1 TMD sequence identity and raft association 88

4.3.3 The role of raft association in desmosome assembly and consequences for disease 90

4.3.4 A newer model of raft-driven segregation of adherens junctions and desmosomes during junction assembly 91

4.3.5 Conclusions 92

4.4 Materials and Methods 93

4.5 Acknowledgements 97

Chapter 5

Future Directions and Conclusions 98

5.1 Introduction 99

5.2 Null model systems for studying desmosomal dynamics 99

5.2.1 Identifying roles for the DSG intracellular domains 100

5.2.2 Null model systems beyond desmogleins 104

5.3 Further exploration of desmosomal dynamics with Dsg1TMD variants 104

5.4 Identifying the role of transmembrane prolines in desmogleins 105

5.5 Considering the possible role of motifs in the desmoglein TMD 109

5.5.1 Lipid-binding motifs 112

5.5.2 Cholesterol-binding motifs 112

5.5.3 TMD-TMD dimerization 113

5.5.4 Structural TMD motifs 115

5.6 Understanding the contribution of palmitoylation towards desmosomal processes 115

5.7 The raft associating mechanism of desmocollin 118

5.8 The role of raft association in segregation of adherens junctions and desmosomes 121

5.8.1 Raft association as a driver of junction segregation 123

5.9 Desmosomes assemble and mediate robust cell-cell adhesion through a hierarchy of molecular interactions 124

5.10 Beyond cell-cell adhesion: the desmosome as a stable molecular platform 126

5.11 Final thoughts and conclusions 127

References 130

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