Dynamic regulation of the endothelial adherens junction Open Access

Nanes, Benjamin Andrew (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/jh343s96j?locale=en
Published

Abstract

Endothelial cells, which line the interior surfaces of blood vessels, must join together to form barriers that are stable enough to resist vascular leak, yet also flexible enough to support the dynamic rearrangements necessary for vascular development and function. This dissertation explores the basic cellular mechanisms allowing endothelial cells to establish cell-cell contacts that properly balance stability and plasticity. Endothelial cell-cell adhesion is mediated through the adherens junction complex, and the strength of these junctions depends on the balance of trafficking of adhesion molecules to and from the membrane.

Vascular endothelial (VE)-cadherin, the principal intercellular adhesion molecule in the endothelium, undergoes constitutive endocytosis and recycling, conferring plasticity to cell-cell junctions. This dissertation identifies a dual-function motif in the VE-cadherin cytoplasmic tail as the key mediator of constitutive VE-cadherin endocytosis. The motif alternately serves as a binding site for p120-catenin (p120), stabilizing the cadherin, or as an endocytic signal, driving internalization of the cadherin. This mechanism allows constitutive VE-cadherin endocytosis to contribute to adherens junction dynamics without causing junction disassembly. Mutation of the constitutive endocytic motif results in a cadherin variant that is both p120-uncoupled and resistant to endocytosis. This mutant potently suppresses a key component of angiogenesis, the collective migration of endothelial cells in response to vascular endothelial growth factor, revealing the importance of constitutive VE-cadherin endocytosis to endothelial function.

While constitutive endocytosis of VE-cadherin is required for junction plasticity, inappropriate loss of endothelial adhesion contributes to disease. This dissertation also identifies a distinct motif that drives induced VE-cadherin endocytosis and pathological junction disassembly associated with the endothelial-derived tumor Kaposi sarcoma. Human herpesvirus 8, which causes Kaposi sarcoma, expresses a ubiquitin ligase, K5, which targets VE-cadherin for ubiquitination at sites within the p120-binding region. K5-mediated ubiquitination of VE-cadherin displaces p120 and drives endocytosis and down-regulation of the cadherin. However, K5-induced VE-cadherin endocytosis does not require the constitutive endocytic motif. Thus, multiple context-dependent signals drive VE-cadherin endocytosis in physiologic and disease states, but p120 binding to the cadherin acts as a master regulator guarding cadherin stability.

Table of Contents

Chapter 1. Finding the balance between stability and plasticity . . . . . . . . 1

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Development of the vascular system . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.1 Vasculogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.2 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.3 Signals guiding vascular patterning . . . . . . . . . . . . . . . . . . . . . . . . 5 1.3.1 Inducing permeability and driving growth . . . . . . . . . . . . . . . . 5 1.3.2 Receptors tune the growth factor response . . . . . . . . . . . . . . . . 6 1.3.3 Establishment of sprout morphology . . . . . . . . . . . . . . . . . . . 7 1.3.4 Steering vessel growth . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.3.5 Promoting vessel stability . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4 The adherens junction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.4.1 Cadherins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.4.2 Structural basis of adhesion . . . . . . . . . . . . . . . . . . . . . . . . 13 1.4.3 Catenins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1.4.4 Links to the cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.4.5 Regulating cadherin stability . . . . . . . . . . . . . . . . . . . . . . . . 16 1.5 Cell-cell adhesion in the endothelium . . . . . . . . . . . . . . . . . . . . . . 17 1.5.1 VE-cadherin in development . . . . . . . . . . . . . . . . . . . . . . . . 17 1.5.2 Response to permeability signals . . . . . . . . . . . . . . . . . . . . . 19 1.6 Disruption of endothelial adhesion in disease . . . . . . . . . . . . . . . . . . 22 1.6.1 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1.6.2 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 1.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Chapter 2. Adherens junction turnover: regulating adhesion through cadherin endocytosis, degradation, and recycling . . . . . . . . . . . . . . . . . . . . 27 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2.2 Cadherin endocytosis in development and disease . . . . . . . . . . . . . . . 29 2.3 Cadherin trafficking pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.3.1 Clathrin-mediated endocytosis . . . . . . . . . . . . . . . . . . . . . . . 33 2.3.2 Endocytic adaptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.3.3 Clathrin-independent endocytic pathways . . . . . . . . . . . . . . . . 35 2.3.4 Recycling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.4 Regulation of cadherin endocytosis by catenins . . . . . . . . . . . . . . . . . 38 2.4.1 p120-catenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.4.2 β-catenin and α-catenin . . . . . . . . . . . . . . . . . . . . . . . . . . 42 2.5 Regulation of cadherin endocytosis and degradation by ubiquitination . . . . 44 2.6 Growth factor signaling and cadherin endocytosis . . . . . . . . . . . . . . . . 46 2.7 Cadherin shedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2.8 Summary and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . 51 Chapter 3. p120-catenin binding masks an endocytic signal conserved in classical cadherins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.2.1 The core p120-binding region of classical cadherins is well conserved . 58 3.2.2 The core p120-binding region of VE-cadherin harbors an endocytic signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.2.3 p120 binding can be uncoupled from control of cadherin endocytosis . 63 3.2.4 p120 occupies the DEE sequence to prevent cadherin endocytosis . . . 69 3.2.5 The core p120-binding region is the primary endocytic signal in VE-cadherin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 3.2.6 VE-cadherin mobility does not require endocytosis . . . . . . . . . . . 71 3.2.7 VEGF-induced endothelial cell migration requires cadherin endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 3.4 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Chapter 4. p120-catenin guards cadherin stability against constitutive and inducible endocytic signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 4.2.1 K5 targets VE-cadherin for ubiquitination and down-regulation . . . . 99 4.2.2 K5 induces VE-cadherin endocytosis . . . . . . . . . . . . . . . . . . . 104 4.2.3 Distinct endocytic motifs drive constitutive- and K5-induced VE-cadherin endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . 105 4.2.4 Constitutive- and K5-induced VE-cadherin endocytosis are functionally separable . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 4.2.5 K5 displaces p120 from VE-cadherin . . . . . . . . . . . . . . . . . . . 110 4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4.4 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Chapter 5. From static linkers to effectors of tissue dynamics . . . . . . . . . . . 125 5.1 Context-dependent signals drive VE-cadherin endocytosis . . . . . . . . . . . 126 5.2 p120 dissociation: Quis custodiet ipsos custodes? . . . . . . . . . . . . . . . . 128 5.3 Understanding catenin role switching . . . . . . . . . . . . . . . . . . . . . . 130 5.4 Cadherin diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 5.5 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Apendix A. Derivation of the fluorescence recovery after photobleaching models 135 A.1 Diffusion-limited recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 A.2 First-order-limited recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 A.3 Model evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

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