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Thesis - David Yeung Complete.pdf (4.28 MB)

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Oligomeric Status of Discoidin Domain Receptor Modulates Collagen Binding, Mechanics, and Receptor Phosphorylation

Yeung, David Alexander
http://rave.ohiolink.edu/etdc/view?acc_num=osu1524185313118309

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biomedical Engineering.
Collagen type I is the most abundant extracellular matrix (ECM) protein found in vertebrates. A number of cell surface receptors and as well as glycoproteins and proteoglycans found in the ECM are known to interact with collagen. The clustering or oligomeric state of these proteins is emerging as an important factor in modulating their interaction with collagen. This thesis attempts to better understand the role of oligomeric status of the collagen receptors, Discoidin domain receptors 1 and 2 (DDR1 and DDR2), in their interaction with collagen. DDRs are widely expressed receptor tyrosine kinases which modulate cells signaling as well as ECM remodeling. The first aim of this thesis (Chapter 2) was to understand how ligand binding impacts the clustering or oligomeric state of DDRs interaction. AFM imaging provided insight on how the extracellular domain (ECD) of DDR1 but not DDR2 was able to cluster upon collagen binding. Live cell assays where the murine osteoblastic cell line, MC3T3-E1, was transfected to express full length fluorescent DDR1 and DDR2 proteins showed that DDR1 clusters upon collagen stimulation whereas DDR2 does not. Further, receptor phosphorylation after collagen binding occurred in DDR1 clusters. On the other hand, DDR2 phosphorylation was found in unique filamentous organizations of the protein. Receptor phosphorylation for both DDR1 and DDR2 was observed several hours post collagen binding, consistent with earlier studies. The second aim of this work (Chapter 3) was to investigate how the oligomeric state of DDR ECD affects their binding to collagen and the structural and mechanical properties of the collagen network. Solid phase binding was used to observe how DDR oligomerization increased their binding affinity towards collagen. AFM and Confocal fluorescence microscopy imaging of collagen gels revealed that DDR2-Fc dimers, and to an even greater extent, DDR2-Fc oligomers reduced the fibril diameter and disrupted the network architecture. Results from turbidity assays supported oligomeric state of DDR2-Fc was a stronger inhibitor of collagen fibrillogenesis. Mechanical properties of the collagen networks formed in the presence of dimeric vs. oligomeric DDR2 ECD were examined using macro and micro rheology (Chapter 4). The rheology performed via parallel plate and optical tweezers respectively showed how DDR2-Fc oligomers increased the shear moduli of collagen gels as well as contributed to a unique strain stiffening pattern not seen in the other samples tested. Our novel active two particle optical tweezer technique showed better agreement with the parallel plate rheology compared to more traditional one particle methods. Finally, we summarize the importance of DDRs and receptor clustering in cell-matrix interactions and scope for future studies (Chapter 5).
Gunjan Agarwal (Advisor)
Heather Powell (Committee Member)
Gregory Lafyatis (Committee Member)
Do-Gyoon Kim (Committee Member)
137 p.
Biomedical Engineering
Collagen, Discoidin Domain Receptor, Rheology, Optical Tweezer, Phosphorylation, Receptor Tyrosine Kinase

Recommended Citations

Citations

  • Yeung, D. A. (2018). Oligomeric Status of Discoidin Domain Receptor Modulates Collagen Binding, Mechanics, and Receptor Phosphorylation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524185313118309

    APA Style (7th edition)

  • Yeung, David. Oligomeric Status of Discoidin Domain Receptor Modulates Collagen Binding, Mechanics, and Receptor Phosphorylation. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1524185313118309.

    MLA Style (8th edition)

  • Yeung, David. "Oligomeric Status of Discoidin Domain Receptor Modulates Collagen Binding, Mechanics, and Receptor Phosphorylation." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524185313118309

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.