Role of the cytoplasmic tail in regulating cadherin trafficking Open Access

Abstract

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

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Cell
Keyword	VE-cadherin Cell Junction Cell Adhesion
Committee Chair / Thesis Advisor	Kowalczyk, Andrew, Emory University
Committee Members	Chen, Ping Helen, Emory University Moberg, Kenneth H, Emory University Koval, Michael H, Emory University Faundez, Victor, Emory University

Last modified

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Role of the cytoplasmic tail in regulating cadherin trafficking ()	2018-08-28 16:23:35 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions