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

Cadwell, Chantel Marie (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/0g354g13m?locale=en
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Abstract

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 mutant ............................................................................... 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

Figure 1. Molecular components of the adherens junction ............................................... 42

Figure 2. Domains of classical cadherins ......................................................................... 43

Figure 3. Homophilic cadherin interactions ...................................................................... 45

Figure 4. Membrane trafficking pathways of cadherins ................................................... 46

Figure 5. Polarized migratory cells ................................................................................... 47

Figure 6. Cadherin treadmilling ........................................................................................ 48

Figure 7. Vascular Development ...................................................................................... 64

Figure 8. Spouting angiogenesis ....................................................................................... 65

Figure 10. Induced dimerization of VE-cadherin inhibits its endocytosis independent of

adhesion ............................................................................................................................ 87

Figure 11. p120-catenin binding is not required for inhibited dimer internalization ........ 89

Figure 12. Ankyrin-G associates with the juxtamembrane domain of VE-cadherin ........ 92

Figure 13. Ankyrin-G inhibits internalization of VE-cadherin ......................................... 93

Figure 14. Ankyrin-G associates with VE-cadherin dimers and does not inhibit

endocytosis of W2 mutant ................................................................................................ 95

Figure 15. Ankyrin-G association is required to inhibit VE-cadherin dimer internalization

........................................................................................................................................... 97

Figure 16. Ankyrin-G regulates adherens junction organization in endothelial cells ....... 99

Figure 17. Model of ankyrin-G mediated inhibition of the internalization of VE-cadherin

dimers .............................................................................................................................. 101

Figure 18. VE-cadherin endocytic mutant requires adhesion to suppress migration ..... 115

Figure. 19 VE-cadherin endocytic mutant requires catenin-binding domain to suppress

migration ......................................................................................................................... 116

Figure 20. VE-cadherin endocytosis is required for Golgi orientation ........................... 118

Figure 21. Endocytosis is required for leading edge accumulation of VE-cadherin ...... 119

Figure 22. Model of VE-cadherin endocytosis during directed, collective cell migration

......................................................................................................................................... 121

Figure 23. Summary and model ...................................................................................... 135


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