Role of the cytoplasmic tail in regulating cadherin trafficking Open Access

Su, Wenji (2017)

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Cell adhesion is essential in many biological processes including embryonic development and disease progression. Dynamic regulation of cadherin-based adhesion is important in modulating cadherin function and adhesion strength. The cytoplasmic tail of classical cadherins acts as a regulatory domain for cadherin activities such as attachment to the cytoskeleton and cadherin turnover, through interactions with a variety of binding partners.

In this dissertation, I focused on two different mechanisms that regulate cadherin trafficking. First, I provided insights into a novel proteolytic event that alters the post-endocytic trafficking itinerary of VE-cadherin. By using a combination of biochemical and fluorescence imaging approaches, I demonstrated that the VE-cadherin tail is cleaved by calpain upon entrance into clathrin-enriched membrane domains before reaching the early endosome, and this cleavage occurs between the juxtamembrane domain and the catenin-binding domain of VE-cadherin. I also found that the cleaved fragment exhibits a higher turnover rate when compared to the full-length cadherin, likely due to a decreased recycling rate implied by reduced colocalization with the recycling markers, as well as increased degradation rate implied by increased colocalization with lysosomal markers.

In addition, I also demonstrated that mutation of a dileucine motif to alanine, which inhibits E-cadherin endocytosis, reduces collective cell migration when measured by a 2-D migration assay, in both normal epithelial cells and lung cancer cells. By disrupting cadherin endocytosis with the dileucine mutation, cancer cell invasion was increased in a 3-D spheroid invasion assay. These results together suggest a context-dependent function of cadherin endocytosis in different biological systems.

In summary, these findings contribute to a better understanding of the role of the cytoplasmic tail in cadherin trafficking and how cadherin trafficking affects cell mobility.

Table of Contents

Chapter 1 Dissertation overview and significance --------------------------------------------- 1

Chapter 2 Overview of adherens junctions ------------------------------------------------------ 7

2.1 Introduction to the adherens junction ----------------------------------------------- 8

2.2 Molecular components of adherens junctions ------------------------------------- 9

2.2.1 Cadherins -------------------------------------------------------------------- 9

2.2.1a Cadherin ectodomain ------------------------------------------ 10

2.2.1b Cadherin cytoplasmic tail ------------------------------------- 12

2.2.2 Other components of the adherens junction --------------------------- 13

2.2.2a p120-catenin ---------------------------------------------------- 14

2.2.2b β-catenin --------------------------------------------------------- 16

2.2.2c Plakoglobin ----------------------------------------------------- 18

2.2.2d Ankyrin ---------------------------------------------------------- 19

2.2.2e α-catenin and actin --------------------------------------------- 20

2.3 Cadherin turnover -------------------------------------------------------------------- 22

2.3.1 Biosynthesis and transportation towards the plasma membrane --- 22

2.3.2 Endocytosis --------------------------------------------------------------- 24

2.3.2a Endocytic motifs in the cadherin tail ------------------------ 26

2.3.3 Recycling and degradation ---------------------------------------------- 30

2.4 Proteolytic processing of cadherin ------------------------------------------------ 32

2.4.1 Overview of cadherins proteolysis ------------------------------------- 32

2.4.2 Proteolytic processing of the cadherin tail ---------------------------- 33

2.4.2 Calpain and cadherins ---------------------------------------------------- 35

2.5 Other post-translational modifications of the cadherin tail --------------------- 38

2.5.1 Phosphorylation ----------------------------------------------------------- 38

2.5.2 Ubiquitination ------------------------------------------------------------- 39

2.5 Concluding remarks ----------------------------------------------------------------- 42

Chapter 3 Proteolytic processing of VE-cadherin tail regulates cadherin trafficking ---- 53

3.1 Introduction --------------------------------------------------------------------------- 54

3.2 Results --------------------------------------------------------------------------------- 57

3.2.1 VE-cadherin is cleaved during endocytosis --------------------------- 57

3.2.2 Endocytosis is required for VE-cadherin cleavage ------------------- 58

3.2.3 VE-cadherin is cleaved at the plasma membrane upon entry into clathrin-enriched domains ------------------------------------------------------ 59

3.2.4 The catenin-binding domain regulates cadherin turnover rates ---- 60

3.2.5 VE-cadherin is cleaved by calpain ------------------------------------- 61

3.3 Discussion ---------------------------------------------------------------------------- 63

3.4 Material and methods --------------------------------------------------------------- 67

Chapter 4 Role of the dileucine motif in E-cadherin endocytosis and cell mobility ----- 89

4.1 Introduction --------------------------------------------------------------------------- 90

4.2 Results --------------------------------------------------------------------------------- 93

4.2.1 Dileucine motif is required for E-cadherin endocytosis ------------- 93

4.2.2 Mutation of the dileucine motif results in reduced collective cell migration rate -------------------------------------------------------------------------------------- 93

4.2.3 Mutation of the dileucine motif results in increased invasive ability of lung cancer cells -------------------------------------------------------------------------------- 94

4.3 Discussion ---------------------------------------------------------------------------- 96

4.4 Material and methods -------------------------------------------------------------- 100

Chapter 5 Dissertation summary and future direction -------------------------------------- 109

5.1 Summary of findings --------------------------------------------------------------- 110

5.2 Remaining questions and future directions ------------------------------------- 113

References ---------------------------------------------------------------------------------------- 121

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