Characterization of tight junction spikes and their role in regulating alveolar barrier function Pubblico
Lynn, Kelly Sabrina (Spring 2021)
Abstract
Regulation of paracellular permeability within tissues is necessary for maintaining proper fluid balance and tissue function. This is especially important in the alveoli of the lungs, where careful tailoring of fluid helps to maintain open airspaces for gas exchange. Chronic alcohol abuse has been linked with leaky lung barrier function, priming it for acute respiratory distress syndrome (ARDS), a serious condition characterized by airspace flooding and widespread inflammation. Cells primarily modulate paracellular permeability through tight junction proteins, particularly transmembrane proteins called claudins. Here, we demonstrate in a rat model that chronic alcohol leads to an increase in claudin-5, which is necessary and sufficient for decreasing barrier function in alveolar epithelial cells (AECs). We further show that claudin-5 disrupts claudin-18 interactions with scaffolding protein ZO-1, suggesting a possible mechanism for alcohol-induced barrier dysfunction. Increased claudin-5 with alcohol was also associated with a rearrangement of tight junctions into spike-like structures perpendicular to cell junction interfaces. These “tight junction spikes” (TJ spikes) appear to be active areas of junction remodeling driven by increased endocytosis of tight junction proteins and form away from pools of β-catenin associated with actin filaments. This suggests a role for adherens junctions in determining the directionality of TJ spike formation. Treatment with the endocytosis inhibitor Dynasore, which targets the actin-binding protein dynamin, significantly reduces the number of TJ spikes and was associated with actin rearrangement into cortical actin. Dynamin-2 was found to colocalize with claudin-18 and ZO-1 at linear junctions but did not appear to localize with β-catenin and TJ spikes. We then used an in situ method of determining barrier function to show that TJ spikes were not sites of increased leak. To begin elucidating possible functions for TJ spikes, we investigated the local claudin-18 proteome using BioID, which showed association with multiple junction proteins including focal adhesion proteins. Of particular note, several proteins involved in signal transduction were biotin-labeled, setting the stage for future work defining potential roles for TJ spikes as signaling platforms.
Table of Contents
CHAPTER 1: INTRODUCTION
Lung anatomy 1
Alveolar epithelium 1
Tight junctions 3
Claudin classification 4
Protein interactions within tight junctions 6
Tight junction recruitment and recycling 8
Claudins in the alveolar epithelium 9
Alcohol and acute respiratory distress syndrome 11
Oxidative Stress Due to Alcohol Exposure 12
Transcription factors associated with lung injury 14
Barrier dysfunction 15
Cytoskeletal interactions and tight junction morphological changes 17
Scope of dissertation 19
Literature cited 23
CHAPTER 2: RUFFLES AND SPIKES: CONTROL OF TIGHT JUNCTION MORPHOLOGY AND PERMEABILITY BY CLAUDINS
Abstract 43
Introduction 44
Ruffled Junctions 46
Roles for claudin/ZO-1 interactions in tight junction ruffling 47
Hypoxia-induced tight junction ruffles 50
Integrin-stimulation by nanostructured surfaces 52
Ruffles formed by mechanical stimulation 53
Tight junction spikes and discontinuities 54
Tight junction spikes as organizers of vesicular traffic 55
Spikes formed in response to chronic alcohol exposure are due to impaired claudin/ZO-1 interactions 57
Roles for claudins in regulating tight junction ultrastructure 58
Summary and future directions 60
Acknowledgements 62
Author contributions 62
Abbreviations 62
Figure 2.1 64
Figure 2.2 65
Figure 2.3 66
Figure 2.4 67
Figure 2.5 68
Figure 2.6 69
Table 2.1 70
Literature cited 71
CHAPTER 3: REGULATION OF CLAUDIN/ZONULA OCCLUDENS-1 COMPLEXES BY HETERO-CLAUDIN INTERACTIONS
Abstract 88
Introduction 88
Results 90
Chronic alcohol alters lung tight-junction permeability. 90
Increased claudin-5 causes increased paracellular leak. 91
Tight junction spikes are associated with barrier disruption. 92
Claudin-5 alters interactions between claudin-18 and ZO-1. 94
A claudin-5 peptide improves alveolar barrier function. 97
Discussion 98
Methods 102
Acknowledgements 113
Author contributions 114
Figure 3.1 115
Figure 3.2 117
Figure 3.3 119
Figure 3.4 121
Figure 3.5 123
Figure 3.6 125
Figure 3.7 127
Literature cited 129
CHAPTER 4: ASYMMETRIC DISTRIBUTION OF DYNAMIN-2 AND β-CATENIN RELATIVE TO TIGHT JUNCTION SPIKES IN ALVEOLAR EPITHELIAL CELLS
Abstract 135
Introduction 136
Materials and Methods 137
Results 144
Morphological classification of tight junction spikes 144
Tight junction spikes are not sites of increased paracellular leak 146
Adherens junctions are asymmetrically opposed to tight junction spikes 147
Dynamin-2 regulates tight junction morphology and function 148
Discussion 150
Acknowledgements 154
Figure 4.1 155
Figure 4.2 157
Figure 4.3 159
Figure 4.4 161
Figure 4.5 163
Figure 4.6 165
Figure 4.7 167
Figure 4.8 169
Figure 4.9 171
Supplemental Figure 4.1 173
Supplemental Figure 4.2 174
Literature Cited 175
CHAPTER 5: IDENTIFICATION OF THE CLAUDIN-18 PROXIMAL PROTEOME USING AN N-TERMINAL BIOTIN LIGASE
Introduction 182
Materials and Methods 183
Results 190
BirA-claudin-18 localizes to cell junctions 190
Evaluation of streptavidin bead elution methods 191
Proteins biotinylated by BirA-claudin-18 192
Discussion 195
Figure 5.1 199
Figure 5.2 200
Figure 5.3 201
Figure 5.4 202
Figure 5.5 203
Table 5.1: Enriched proteins tagged by biotin ligase fused to claudin-18. 204
Table 5.2: Tight junction, adherens junction, and focal adhesion proteins tagged by biotin ligase fused to claudin-18. 209
Table 5.3: Selected signal transduction proteins tagged by biotin ligase fused to claudin-18. 211
Table 5.4: Proteosome and protein processing in endoplasmic reticulum proteins tagged by biotin ligase fused to claudin-18. 215
Table 5.5: Endocytosis and phagocytosis proteins tagged by biotin ligase fused to claudin-18. 217
Literature Cited 219
CHAPTER 6: DISCUSSION – CONCLUSION AND FUTURE DIRECTIONS 229
Alcohol-induced changes in tight junction protein interactions 230
Tight junction spikes as separate sites of activity 232
Dynamin-2-actin bundling and tight junction spike formation 233
Asymmetrical formation of tight junction spikes and β-catenin 234
Tight junction spikes and localized permeability 236
New technologies for tight junction research 236
Therapeutic outlook 237
Summary 238
Literature Cited 240
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