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theses

Adherens junctions support strong cell-cell adhesions to create tissues. The core of the adherens junction protein complex consists of three parts: 1) an adhesion transmembrane receptor called E-cadherin, 2) the plaque proteins: p120-catenin, β-catenin and α-catenin, 3) and actin, to anchor these adhesive components (e.g. E-cadherin and plaque proteins). The E-cadherin ectodomain binds to an identical molecule on the surface of an adjacent cell. The E-cadherin cytoplasmic domain is required for the plaque proteins to bind, which in turn bind the adhesion complex to the actin cytoskeleton. The events induced by E-cadherin binding leads to the development of tissue with well-defined apical, lateral and basal cell zones. Previously, it was demonstrated E-cadherin overexpression rescues permeability barrier in tissues assembled from MDCK cells where β-actin monomer synthesis is partially delocalized. β-actin mRNA contains a nucleotide sequence in its 3’UTR region called the zipcode; and the translational regulator zipcode binding protein-1, ZBP1, recognizes this sequence. Both the zipcode sequence and ZBP1 are essential for targeting and translational regulation of the β-actin transcript. Additionally, a wide range of tyrosine kinases are present at cell-cell contact zones. E-cadherin homophilic binding leads to Src activation, a non-receptor tyrosine kinase, at cell-cell contact zones. Active Src is known to phosphorylate ZBP1 which is bound to the β-actin mRNA zipcode nucleotide sequence, initiating β-actin monomer synthesis. In this investigation using Madin-Darby Canine Kidney (MDCK) cells, I demonstrate E-cadherin overexpression in cells partially delocalizing β-actin monomer synthesis rescues adherens junction assembly. I also show E-cadherin homophilic binding and protein expression regulates the spatial localization of β-actin monomer synthesis at cell-cell contact zones during de novo adherens junction formation. Furthermore, inhibiting E-cadherin function or protein expression decreases cell-cell contact localized active Src. Interestingly, inhibiting E-cadherin endocytosis in MDCK cells with partial β-actin monomer synthesis delocalization during de novo cell-cell contact rescues tissue permeability barrier and adherens junction assembly; both of these rescues are prevented when the β-actin mRNA zipcode is masked using antisense oligonucleotides. Taken together, these data reveal the relationship between E-cadherin, active Src and β-actin monomer synthesis at cell-cell contact during de novo adherens junction assembly. Also, these data support a model where E-cadherin mediates spatially regulated β-actin protein expression during de novo adherens junction assembly through the mRNA zipcode/ZBP1 pathway.

ETD_8957

Ph.D.

Includes bibliographical references

by Lisette A. Cruz

doi:10.7282/t3-y6b9-fx10

ETD doctoral

The author owns the copyright to this work.