Cadherin endocytosis: mechanisms of regulation and implications for endothelial cell migration Open Access

Cadwell, Chantel Marie (2016)

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Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Thus, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. VE-cadherin is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. Many proteins interact with the cadherin to regulate expression at the plasma membrane, including catenins. p120-catenin binds to the cytoplasmic tail of the cadherin and stabilizes it at the plasma membrane. In addition, VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which increases the adhesive strength of the junction. However, the relationship between cadherin endocytosis and cadherin adhesive interactions is still not fully understood. In addition, the role of VE-cadherin endocytosis during developmental processes, such as collective cell migration, is not known. Here, we provide insight into the dynamic relationship between adhesion and endocytosis. We find that cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. Importantly, we find that ankyrin-G, a cytoskeletal adaptor protein, associates with and inhibits the endocytosis of VE-cadherin cis dimers independent of p120 binding. Ankyrin-G binding is important for junctional organization. Depletion of ankyrin-G results in disrupted localization of junctional proteins, including VE-cadherin, p120, and β-catenin. In addition, we define a role for VE-cadherin endocytosis during directed, collective cell migration. Previously, our lab found that mutation of specific amino acids in the VEcadherin cytoplasmic tail prevents endocytosis of the cadherin and inhibits collective cell migration. Here we report that the VE-cadherin endocytic mutant inhibits collective cell migration through a mechanism that involves adhesion and cytoskeletal linkage. Furthermore, we have found that VE-cadherin endocytosis is required for leading edge accumulation of the cadherin and for Golgi orientation at the wound edge, processes which polarize cells and promote directed cell migration. Understanding the mechanisms that regulate cadherin adhesion and endocytosis provides insight into processes that are important for development and disease pathology.

Table of Contents

Chapter 1: Dissertation Overview and Significance. 1

Chapter 2: Molecular mechanisms of cadherin-based junctions. 7

2.0 Introduction to cadherin based junctions. 8

2.1 Molecular components of Adherens junctions. 8

2.1.1 Cadherins. 9

2.1.2 Catenins: structure and function. 11

2.2 Mechanisms of cadherin based adhesion. 16

2.2.1 Trans interaction. 16

2.2.2 X-dimer. 17

2.2.3 Cis interaction. 18

2.2.4 Cadherin clusters. 19

2.2.5 Junction assembly. 20

2.3 Cadherin trafficking. 21

2.3.1 Trafficking pathways. 21

2.3.2 Regulation of cadherin fate. 22

2.4 Regulation of cadherin endocytosis. 23

2.4.1 Cadherin homophilic interactions. 24

2.4.2 Catenins. 25

2.4.3 Regulation of cadherin endocytosis through adaptor proteins. 27

2.4.4 Ubiquitin. 29

2.4.5 Growth Factors. 31

2.5 Adherens junctions in cell migration. 33

2.5.1 Directed, collective cell migration. 33

2.5.2 Migratory polarization. 35

2.6 Concluding Remarks. 39

Chapter 3: Adherens junctions of the vascular endothelium. 49

3.0 Adherens junctions of the vascular endothelium. 50

3.1 Vascular development. 50

3.2 Endothelial cell migration during angiogenesis. 53

3.3 VE-cadherin in vascular development. 55

3.4 The role of VE-cadherin in cell polarity. 58

3.5 VE-cadherin endocytosis and migration. 59

3.6 Endothelial junctions in vascular disease. 61

3.7 Summary. 63

Chapter 4: Ankyrin-G inhibits endocytosis of cadherin dimers. 66

4.0 Introduction. 67

4.1 Results. 69

4.1.1 Induced dimerization of VE-cadherin inhibits endocytosis independent of adhesion. 69

4.1.2 p120-catenin binding is not required for inhibited endocytosis of VE-cadherin dimers. 70

4.1.3 VE-cadherin colocalizes with and co-immunoprecipitates with Ankyrin-G. 71

4.1.4 Ankyrin-G inhibits internalization of VE-cadherin independent of p120-catenin binding. 72

4.1.5 Ankyrin-G selectively associates with dimerized VE-cadherin and does not inhibit endocytosis of W2 muta 73

4.1.6 Ankyrin-G association is required to inhibit VE-cadherin dimer internalization. 74

4.1.7 Ankyrin-G regulates adherens junction organization in endothelial cells. 74

4.2 Discussion. 75

4.3 Methods and Materials. 80

Chapter 5: Cadherin endocytosis regulates cell polarity during directed collective cell migration. 102

5.0 Introduction. 103

5.1 Results. 105

5.1.1 Cadherin endocytosis, adhesion and cytoskeletal linkages cooperate to regulate cell migration. 105

5.1.2 VE-cadherin endocytosis is required for Golgi orientation toward the wound edge in endothelial cells. 106

5.1.3 VE-cadherin endocytosis is required for leading edge localization of adherens junction proteins. 107

5.2 Discussion. 108

5.4 Materials and Methods. 110

Chapter 6: Dissertation summary and future directions. 123

6.0 Dissertation Summary. 124

6.1 Future Directions. 125

Chapter 7: References. 136

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