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Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins.

ABSTRACT: Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.

SUBMITTER: Ichikawa T

PROVIDER: S-EPMC9122943 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins.

Ichikawa Takehiko T Wang Dong D Miyazawa Keisuke K Miyata Kazuki K Oshima Masanobu M Fukuma Takeshi T

Communications biology 20220520 1

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. I ...[more]

PMID: 35595960

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Project description:The influence of surface topography on protein conformation and association is used routinely in biological cells to orchestrate and coordinate biomolecular events. In the laboratory, controlling the surface curvature at the nanoscale offers new possibilities for manipulating protein-protein interactions and protein function at surfaces. We have studied the effect of surface curvature on the association of two proteins, α-lactalbumin (α-LA) and β-lactoglobulin (β-LG), which perform their function at the oil-water interface in milk emulsions. To control the surface curvature at the nanoscale, we have used a combination of polystyrene (PS) nanoparticles (NPs) and ultrathin PS films to fabricate chemically pure, hydrophobic surfaces that are highly curved and are stable in aqueous buffer. We have used single-molecule force spectroscopy to measure the contour lengths Lc for α-LA and β-LG adsorbed on highly curved PS surfaces (NP diameters of 27 and 50 nm, capped with a 10 nm thick PS film), and we have compared these values in situ with those measured for the same proteins adsorbed onto flat PS surfaces in the same samples. The Lc distributions for β-LG adsorbed onto a flat PS surface contain monomer and dimer peaks at 60 and 120 nm, respectively, while α-LA contains a large monomer peak near 50 nm and a dimer peak at 100 nm, with a tail extending out to 200 nm, corresponding to higher order oligomers, e.g. trimers and tetramers. When β-LG or α-LA is adsorbed onto the most highly curved surfaces, both monomer peaks are shifted to much smaller values of Lc. Furthermore, for β-LG, the dimer peak is strongly suppressed on the highly curved surface, whereas for α-LA the trimer and tetramer tail is suppressed with no significant change in the dimer peak. For both proteins, the number of higher order oligomers is significantly reduced as the curvature of the underlying surface is increased. These results suggest that the surface curvature provides a new method of manipulating protein-protein interactions and controlling the association of adsorbed proteins, with applications to the development of novel biosensors.

Dataset Information

Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins.

Publications

Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins.

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