Influence of the extracellular domain size on the dynamic behavior of membrane proteins

Gurdap, Cenk Onur; Wedemann, Linda; Sych, Taras; Sezgin, Erdinç

doi:10.1016/j.bpj.2022.09.010

Cited by 16 publications

(20 citation statements)

References 61 publications

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“…Using targeted antigen probes, we expand our spatial resolution laterally, in the plane of the membrane, measuring differences in crowding between plasma membrane domains on live tumor cells. Our findings support the hypothesis that raft-like domains of native membranes exclude proteins with bulky extracellular domains, consistent with the findings of Gurdap et al on GPMVs [17]. Our simple IgG binding assay to probe spatial heterogeneities on native cell membranes suggests an important role of structural complexities on glycocalyx organization.…”

Section: Introductionsupporting

confidence: 92%

Antibody binding reports spatial heterogeneities in cell membrane organization

Arnold

Takatori

2022

Preprint

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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 heterogeneities 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.

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Section: Introductionsupporting

confidence: 92%

Antibody binding reports spatial heterogeneities in cell membrane organization

Arnold

Takatori

2022

Preprint

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“…Recent studies have shown that the glycocalyx can also attenuate the binding of viruses and lectins to cell surface receptors (5)(6)(7). Surface crowding also alters protein mobility and sorting (8), as well as membrane channel gating (9). However, quantitative methods to obtain a detailed, mechanistic understanding of plasma membrane density and the biophysical interactions that govern macromolecular binding on live cell surfaces are lacking.…”

Section: Introductionmentioning

confidence: 99%

Engineered molecular sensors of cell surface crowding

Takatori

Son

Lee

et al. 2022

Preprint

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Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2-20 fold in live cells compared to a bare membrane surface, resulting in a cell surface osmotic pressure opposing binding of 1 - 4 kPa. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface osmotic pressure may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

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“…In light of this and taking into account that HuTu80 cells carry less HSPGs but slightly more CAR on their cell surface than A549 cells (data not shown), EAdV may be slowed by multivalent interactions with HSPGs on A549 and have a surface sampling advantage leading to faster internalization in HuTu80 cells. This hypothesis is also supported by the finding that large extracellular domains, like HSPGs, have smaller diffusion coefficients on giant luminal vesicles (45). To validate this hypothesis, virus particles with short or long fibers only, would be valuable tools to assess the contribution of known receptors to the entry kinetics and diffusion behaviour.…”

Section: Discussionmentioning

confidence: 80%

Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells

Becker

Dorma

Conca

et al. 2023

Preprint

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Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. Unlike other adenoviruses, EAdVs exhibit hitherto a strict tropism for gastrointestinal tissues with, to date, unknown infection mechanism and target cells. In this study, we turn to potentially limiting host factors by comparison of EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking and transmission electron microscopy. Our analyses highlight kinetic advantages in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understand pathogenesis and develop new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a physiologically relevant cell line.Author SummaryEnteric adenoviruses have historically been difficult to grow in cell culture, which resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

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Influence of the extracellular domain size on the dynamic behavior of membrane proteins

Cited by 16 publications

References 61 publications

Antibody binding reports spatial heterogeneities in cell membrane organization

Antibody binding reports spatial heterogeneities in cell membrane organization

Engineered molecular sensors of cell surface crowding

Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells

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