Abstract
The mammalian plasma membrane regulates cellular interactions with the extracellular environment through a dense assembly of membrane glycoproteins and glycolipids. Although the spatial organization of the cell surface glycocalyx is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma membrane, we currently do not have the methods to quantify the spatial hetero-geneities on live cell surfaces. In this work, we engineer molecular antigen sensors that act as a direct reporter of live cell surface crowding heterogeneities with nanometer spatial resolution. By quantifying the effective binding affinity of IgG monoclonal antibodies to our antigen sensors on reconstituted and live cells, we provide a biophysical understanding of the molecular-to-mesoscale spatial organization of the glycocalyx. We find that the antigen location strongly influences the IgG binding on the red blood cell membrane, with the strongest gradients occurring within a few nanometers of the membrane. We develop an analytical theory and coarse-grained molecular dynamics simulations to corroborate our results, and combine surface proteomics with our simulations and theory for an in-silico reconstruction of the red blood cell surface. We also show that the effective binding affinity above raft-like domains is much higher than that of the bulk membrane on human cancer cells, suggesting that raft-like domains exclude membrane proteins with bulky extracellular domains. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may facilitate monoclonal antibody design and provide a mechanistic understanding of plasma membrane biophysical organization.
Significance Statement The mammalian cell surface is decorated with a protective “shield” of membrane proteins and sugars that enables cells to mediate interactions with the extracellular environment. The precise spatial distribution of the proteins and sugars are critical for cell function, but we currently do not have the techniques to quantify the spatial heterogeneities on the cell membrane surface. In this work, we develop a method to quantify the spatial organization of the cell surface by engineering molecular antigen sensors that act as a direct reporter of local macro-molecular crowding.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
D.P.A. and S.C.T. conceived of the study; all authors designed research; D.P.A. performed experiments; Y.X. performed simulations; S.C.T. supervised the study; and all authors wrote the paper.
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