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Title:Regulation of cadherin adhesion at intercellular junctions
Author(s):Shashikanth, Nitesh
Director of Research:Leckband, Deborah
Doctoral Committee Chair(s):Leckband, Deborah
Doctoral Committee Member(s):Martinis, Susan; Brieher, William; Tajkhorshid, Emad
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Cadherin
Kinetics
Abstract:Adhesion proteins maintain cell-cell interactions, which are critical for tissue formation and the hierarchical organization of all multicellular organisms, and among them, cadherins are the major transmembrane cell-cell adhesion proteins in all vertebrate tissues. Regulation of cadherin mediated adhesion at cell-cell junctions is crucial to our understanding of development and disease. This thesis focuses on the regulation of cadherin adhesion, which can be influenced by its extracellular domain interactions, ligand or antibody binding, post translational modifications, or inside out signaling from cytoplasmic binding proteins. In this thesis, micropipette-based adhesion frequency measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that appears to reflect both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. These kinetic measurements were used to assess the impact of confinement within narrow adhesion zones on the assembly of intercellular adhesions. Specifically, a unique kinetic signature suggested the formation of lateral interactions that were not detected in solution binding assays. Mutations postulated to disrupt lateral cadherin association altered the kinetic signature, but did not affect cadherin binding affinity. Perturbed kinetics further correlated with altered cadherin clustering at cell-cell junctions, wound healing dynamics, and paracellular permeability. Adhesion frequency measurements were used to demonstrate the allosteric regulation of cadherin adhesive function. In this thesis, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that activating anti-E-cadherin monoclonal antibodies or the dephosphorylation of a cytoplasmic binding partner, p120 catenin, increased the homophilic binding affinity of E-cadherin on Colo 205 cells. Further studies of Colo 205 cells demonstrated that four treatments, which similarly altered p120 catenin phosphorylation resulted in quantitatively similar enhancement in E-cadherin affinity. Using this approach, I further investigated the effect of N-linked and O-linked glycosylation on E-cadherin activity and function. Results revealed that, contrary to the influence of glycosylation on N-cadherin function, N-glycosylation of E-cadherin in the EC4 and EC5 domains negatively regulated cadherin adhesion, by altering binding kinetics and clustering at cell-cell junctions. This suggests the influence of N-glycosylation depends on its position in the cadherin ectodomain. In conclusion, this dissertation describes studies which elucidated different mechanisms regulating cadherin adhesive function. Results showed that cadherin binding is regulated by its ectodomain interactions at cell-cell junctions, by glycosylation, and by allosteric inside-out signaling. These findings were enabled by the adhesion frequency measurements, which enabled quantitative assessment of cadherin binding function, in the native context of the cell membrane and cytosolic binding partners.
Issue Date:2016-06-16
Type:Thesis
URI:http://hdl.handle.net/2142/93006
Rights Information:© 2016 Nitesh Shashikanth
Date Available in IDEALS:2016-11-10
Date Deposited:2016-08


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