Date of Award

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Physiology

Research Advisor

Radhakrishna Rao, Ph.D.

Committee

Ramareddy V. Guntaka, Ph.D. David R. Nelson, Ph.D. Michael R. Taylor, Ph.D. Christopher W. Waters, Ph.D.

Keywords

Epithelium, Hot Spot, Occludin, Phophorylation, Regulation, Tight Junctions

Abstract

Tight junctions (TJ) constitute the primary component of epithelial barrier function, a disruption of which is involved in the pathogenesis of many gastrointestinal, pulmonary and renal diseases. Occludin is the major transmembrane protein of TJ, a deletion of which leads to a complex phenotype including chronic inflammation in several epithelial tissues of occludin deficient mice and poor TJ integrity in epithelial cell lines. Its down regulation was seen in Crohn’s disease, tumors of the colon, brain, endometrium and breast cancer. Occludin is also known to be a target that enables Hepatitis C Virus infection and bacterial pathogenesis. But the specific function of occludin in TJ remains unknown. Numerous studies implicated that occludin phosphorylation plays a crucial role in TJ regulation. Based on our previous studies, we have identified a conserved phosphorylation hotspot in the C-terminal domain of occludin, called occludin regulatory motif (ORM), which has a potential to confer dynamic properties to occludin. The present study was designed to determine the function of ORM in TJ regulation. Stable, occludin-deficient MDCK cell line (OD-MDCK) that expresses wild type occludin (OclWT) or a deletion mutant occludin (OclDM) that lacked ORM were developed. Confocal imaging and immunoprecipitation analyses showed that absence of ORM does not prevent occludin localization and interaction with ZO-1 at TJ. However, FRAP analysis showed that mobile fraction of occludin is reduced by the absence of ORM, suggesting the role of ORM in occludin dynamics. Also, Ca2+ depletion-induced disruption of TJ, adherens junctions and cytoskeleton and barrier dysfunction were attenuated in the absence of ORM. Mutation anlaysis of ORM phosphosites Y 398, Y 402, T403 and T404, revealed that ORM-mediated phosphorylation events determine the dynamic properties of TJ. Then the role of ORM-mediated protein interactions in TJ modulation was investigated. A synthetic peptide analogous to occludin regulatory region (ORP), which enhanced TJ assembly and attenuated radiation-induced barrier dysfunction in Caco-2 cells, was used as bait to pull down proteins that interact with ORM. Proteomic analyses and pull down assays using ORP and GST tagged occludin C-terminus identified that MAP7 (an epithelial specific microtubule associated protein) interacts with ORM in a tyrosine phosphorylation-dependent mechanism. Furthermore, MAP7 binding to occludin in Caco-2 cells was elevated by treatment with osmotic stress or dextran sodium sulfate, both known to increase occludin Tyr-phosphorylation. These data demonstrated that MAP7 interacts with ORM and mediates occludin redistribution during TJ disruption. In conclusion ORM confers dynamic properties to TJ via its phosphorylation dependent protein-protein interactions. The findings in my project establish the functional significance of ORM in TJ dynamics and regulatory mechanisms associated with TJ modulation. Identification of proteins that interact with ORM provides new therapeutic targets in the treatment of diseases associated with occludin depletion and epithelial barrier dysfunction.

ORCID

http://orcid.org/0000-0001-5618-3967

DOI

10.21007/etd.cghs.2016.0415

Comments

Two year embargo expires October 2018.

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