All the small things: Utilizing a nanoparticle platform to examine apical integrin regulation of the epithelial barrier Open Access
Peterson, Raven (Summer 2021)
Abstract
Epithelial cells form selectively permeable barriers that compartmentalize internal and external microenvironments and regulate the movement of water, ions, and other solutes critical for maintaining homeostasis. Tight junctions, comprised of transmembrane domain containing proteins (Ig superfamily, MarvelD, and claudins), and scaffold proteins (including the Zonula Occludens (ZO) family) are the major regulators of permeability forming the apical/basolateral barrier. Evidence has emerged that integrins, particularly those localized to the apical surface, play a role in regulating epithelial barrier morphology and function. In order to determine integrin specific contributions to the regulation of the epithelial barrier, we utilized a derivatized polymeric nanoparticle platform conjugated with anti-integrin antibodies to target apically localized integrins in human intestinal epithelial cells. We used immunofluorescence imaging of tight junction proteins and assessed barrier function by measuring flux of ions and small tracer molecules. We found that low aspect ratio nanowires conjugated with anti-integrin antibodies recognizing a closed conformation induced disparate effects on tight junction proteins, where these nanowires stimulated claudin-2, claudin-4, and ZO-1 to assume a ruffled morphology while claudin-1 and claudin-7 remained linear. These anti-closed integrin nanowires also increased permeability to ions and whole IgG, but not calcein. It is possible that these changes were driven by the actin cytoskeleton, as anti-closed integrin nanowires stimulated cortical F-actin localization and increased talin localization to cell/cell contacts. When low aspect ratio nanowires were conjugated with activating anti-integrin antibodies recognizing an extended conformation, they increased the linearity of ZO-1, had no effect on claudin morphology, and decreased permeability to ions. Particle geometry was also critical, since we found that anti-closed integrin antibody conjugated microspheres had little impact on tight junction morphology and ion permeability but activating anti-integrin antibody conjugated microspheres induced ZO-1 ruffling and caused biphasic changes in ion permeability. These observations support a role for integrins in regulating epithelial barrier function and suggests that anti-integrin nanoparticles may provide a tunable platform for regulating tight junction permeability.
Table of Contents
List of figures............................................................................................................................................................xv
List of tables............................................................................................................................................................xvii
Abbreviations.........................................................................................................................................................xviii
Chapter 1: Introduction...............................................................................................................................................1
1.1 The epithelial barrier................................................................................................................................................1
1.2 Scope of the dissertation..........................................................................................................................................2
References....................................................................................................................................................................5
Chapter 2: Ruffles and spikes: Control of tight junction morphology and permeability by claudins..........................7
2.1 Abstract..................................................................................................................................................................7
2.2 Introduction............................................................................................................................................................8
2.3 Ruffled junctions....................................................................................................................................................10
2.3.1 Roles for claudin/ZO-1 interactions in tight junction ruffling..................................................................................12
2.3.2 Hypoxia induced tight junction ruffles...................................................................................................................15
2.3.3 Integrin stimulation by nanostructured surfaces....................................................................................................17
2.3.4 Ruffles formed by mechanical stimulation.............................................................................................................18
2.4 Tight junction spikes and discontinuities.................................................................................................................19
2.4.1 Tight junction spikes as organizers of vesicular traffic............................................................................................21
2.4.2 Spikes formed in response to chronic alcohol exposure are due to impaired claudin/ZO-1 interactions......................23
2.4.3 Roles for claudins in regulating tight junction ultrastructure..................................................................................24
2.5 Summary and future directions...............................................................................................................................26
References..................................................................................................................................................................37
Chapter 3: Above the matrix: Functional roles for apically localized integrins .......................................................57
3.1 Abstract................................................................................................................................................................57
3.2 Introduction..........................................................................................................................................................57
3.2.1 Integrin Structure................................................................................................................................................58
3.2.2 Sensing and manipulating integrin activation using monoclonal antibodies ............................................................59
3.2.3 Roles for divalent ions and disulfide bonds in integrin conformation.......................................................................60
3.3 Function of apical integrins....................................................................................................................................62
3.3.1 Reproduction and control of cell phenotype..........................................................................................................62
3.3.2 Apical integrins as bacterial receptors..................................................................................................................64
3.3.3 Regulation of epithelial barrier function...............................................................................................................66
3.3.4 Regulation of cell migration................................................................................................................................67
3.3.5 Mechanosensing.................................................................................................................................................68
3.4 Summary and future directions...............................................................................................................................70
References..................................................................................................................................................................76
Chapter 4: Apical integrins as a switchable target to regulate the epithelial barrier................................................89
Abstract......................................................................................................................................................................89
4.1 Introduction..........................................................................................................................................................89
4.2 Materials and Methods...........................................................................................................................................92
4.2.1 Fabrication and conjugation of nanowires.............................................................................................................92
4.2.2 Cell culture.........................................................................................................................................................92
4.2.3 Immunofluorescence...........................................................................................................................................93
4.2.4 Barrier function assays.........................................................................................................................................95
4.3 Results..................................................................................................................................................................96
4.3.1 Multimeric engagement of apical integrin β1 by AIIB2 induces tight junction ruffling...............................................96
4.3.2 The effect of targeting apical integrin β1 subunits on barrier function depends on epitope and valency......................99
4.3.3 Effect of targeting apical integrin β1 subunits on actin depend on valency.............................................................100
4.4 Discussion...........................................................................................................................................................102
References................................................................................................................................................................115
Chapter 5: Impact of anti-integrin particle geometry on tight junctions................................................................122
Abstract....................................................................................................................................................................122
5.1 Introduction........................................................................................................................................................123
5.2 Materials and Methods.........................................................................................................................................124
5.2.1 Fabrication and conjugation of nanowires...........................................................................................................124
5.2.2 Cell culture.......................................................................................................................................................125
5.2.3 Immunofluorescence.........................................................................................................................................125
5.2.4 Barrier function.................................................................................................................................................126
5.3 Results................................................................................................................................................................127
5.3.1 Effects of targeting apical integrins on junction morphology is dependent on nanoparticle aspect ratio..................127
5.3.2 Role of aspect ratio on the effects of anti-integrin particles on epithelial barrier structure.....................................128
5.3.3 Apical integrin conformation state and interaction with nanoparticles.................................................................128
5.3.4 Aspect ratio and integrin conformation state control how apical integrins regulate epithelial barrier function........130
5.4 Discussion..........................................................................................................................................................131
References...............................................................................................................................................................141
Chapter 6: Practical issues related to applications of anti-integrin nanowires......................................................144
Abstract..................................................................................................................................................................144
6.1 Introduction.......................................................................................................................................................145
6.2 Materials and Methods........................................................................................................................................147
6.2.1 Fabrication and conjugation of nanowires..........................................................................................................147
6.2.2 Caco-2 cell culture............................................................................................................................................147
6.2.3 NhBE cell culture..............................................................................................................................................147
6.2.4 Glutathione competition...................................................................................................................................148
6.2.5 Immunofluorescence........................................................................................................................................148
6.2.6 Barrier function................................................................................................................................................149
6.3 Results................................................................................................................................................................149
6.3.1 Duration of nanowire mediated changes of epithelial barriers...............................................................................149
6.3.2 Reversibility of anti-integrin nanowire/cell interactions........................................................................................151
6.3.3 The effects of AIIB2 nanowires are abrogated by MMP-2 inhibitors........................................................................152
6.3.4 Targeting apical integrins in upper airway epithelial cells......................................................................................153
6.4 Discussion............................................................................................................................................................154
References.................................................................................................................................................................165
Chapter 7: Conclusions and future directions..........................................................................................................170
7.1 Overview of findings and significance.....................................................................................................................170
7.2 Future directions...................................................................................................................................................172
7.2.1 Integrins............................................................................................................................................................172
7.2.2 The nanoparticle platform itself...........................................................................................................................174
7.2.3 Tension..............................................................................................................................................................176
References..................................................................................................................................................................179
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