Molecular Resolution Mapping of Erythrocyte Cytoskeleton by Ultrastructure Expansion Single‐Molecule Localization Microscopy. Issue 2 (21st December 2022)

• Record Type: Journal Article
• Authors: Hou, Mengdi; Xing, Fulin; Yang, Jianyu; Hu, Fen; Pan, Leiting; Xu, Jingjun
• Is Part Of: Small methods. Volume 7:Issue 2(2023)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-12-21
• Subjects: biconcave shape -- cytoskeletons -- erythrocytes -- expansion microscopy -- super‐resolution microscopy; Nanotechnology -- Methodology -- Periodicals; Nanotechnology -- Periodicals; Periodicals; 620.5028
• DOI: 10.1002/smtd.202201243
• Languages: English
• ISSNs: 2366-9608

Abstract

Abstract: The combination of expansion microscopy and single‐molecule localization microscopy has the potential to approach the molecular resolution. However, this combination meets challenges due to the hydrogel shrinkage in the presence of imaging buffer. Here, a method of ultrastructure expansion single‐molecule localization microscopy (U‐ExSMLM) based on skillfully adhering the gel onto poly‐l ‐lysine (pLL)‐coated coverslip is developed to prevent lateral shrinkage of the hydrogel. U‐ExSMLM is then applied to dissect the membrane cytoskeleton organization of human erythrocytes at molecular resolution. The resolved nanoscale spatial distributions of cytoskeleton proteins, including the N/C‐termini of β‐spectrin, protein 4.1, and tropomodulin, show good agreement with the acknowledged model of erythrocyte cytoskeleton structure, demonstrating the reliability of U‐ExSMLM. Furthermore, the concentration of pLL is adjusted to preserve the physiological biconcave morphology of erythrocytes, and it is found that the spectrin cytoskeleton in the dimple regions has lower density and larger length than that in the rim regions, which provides the direct evidence for cytoskeleton asymmetry in human erythrocytes. Therefore, the integrated method offers future opportunities to study the ultrastructure of membrane cytoskeleton at molecular resolution. Abstract : The combination of expansion microscopy and single‐molecule localization microscopy meets difficulty of gel shrinkage in the presence of imaging buffer. Here, Hou et al. develop an ultrastructure expansion single‐molecule localization microscopy (U‐ExSMLM) based on application of poly‐l ‐lysine (pLL) to prevent lateral shrinkage of the gel. The integrated method reaches the resolution of ≈6 nm and resolves the asymmetry of human erythrocyte cytoskeleton at molecular resolution.