INVESTIGATION OF THE IMPACT OF GLYCOCALYX CROWDING ON MOLECULAR BEHAVIORS OF CELL SURFACE RECEPTORS
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Macromolecular crowding on biological membranes can influence membrane protein diffusion, aggregation, and biomolecular interactions. Cancer cells remodel their sugary cell surface coating -the cell glycocalyx- by overexpressing large glycosaminoglycans and mucin glycoproteins, which may contribute to crowding on the cell surface. While altered Receptor Tyrosine Kinase (RTK) activity is strongly associated with many types of cancer, our understanding of how glycocalyx crowding impacts RTK activation remains limited. Current work focuses on biochemical interactions among RTK and other non-RTK membrane proteins but overlooks the profound effect of the crowded environment itself. To address this, there is a need for the stable and controllable synthesis of customized mucin glycoproteins to engineer specific states of macromolecular crowding on the cell surface. Here, I established a modular library of sequence-specific mucins with varying length and extent of glycosylation. The customized mucin glycoproteins help reveal the sequence-specific effects on O-linked mucin glycosylation. Furthermore, fusion of mucin ectodomains to suitable membrane anchors and adoption of inducible expression systems permit the control of the density and thickness of the mucin-rich glycocalyx. I then utilize a FRET-based crowding sensor to demonstrate that cell glycocalyx is such a crowded environment as the cell cytosol. The cell surface receptors, which are embedded in the cell glycocalyx, exhibit crowding-induced behaviors. Taking Epithermal Growth Factor Receptor (EGFR) as an example, I find that diffusivity of EGFR decreases upon increased mucin crowding. Single molecule behavior analysis indicates that on the crowded cell surface, EGFR diffusion is slower and shows correlations in velocities consistent with crowding effects. Moreover, glycocalyx crowding promotes EGFR oligomerization. Together, these studies establish a new physical role for the glycocalyx in cell surface receptor activation.
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176 pages
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2020-08
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EGFR; glycocalyx; glycosylation; macromolecular crowding; mucin; synthetic biology
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Paszek, Matthew J.
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Cosgrove, Ben
Baird, Barbara A.
Baird, Barbara A.
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Biophysics
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Ph. D., Biophysics
Degree Level
Doctor of Philosophy
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Government Document
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Attribution 4.0 International
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dissertation or thesis