Super-Resolution Microscopy Reveals the Native Ultrastructure of the Erythrocyte Cytoskeleton

Leiting Pan,Rui Yan,Wan Li,Ke Xu

doi:10.1016/j.celrep.2017.12.107

Leiting Pan, Rui Yan + Show 2 more

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https://doi.org/10.1016/j.celrep.2017.12.107

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Abstract

The erythrocyte cytoskeleton is a textbook prototype for the submembrane cytoskeleton of metazoan cells. While early experiments suggest a triangular network of actin-based junctional complexes connected by ∼200-nm-long spectrin tetramers, later studies indicate much smaller junction-to-junction distances in the range of 25-60nm. Through super-resolution microscopy, we resolve the native ultrastructure of the cytoskeleton of membrane-preserved erythrocytes for the N and C termini of β-spectrin, F-actin, protein 4.1, tropomodulin, and adducin. This allows us to determine an ∼80-nm junction-to-junction distance, a length consistent with relaxed spectrin tetramers and theories based on spectrin abundance. Through two-color data, we further show that the cytoskeleton meshwork often contains nanoscale voids where the cell membrane remains intact and that actin filaments and capping proteins localize to a subset of, but not all, junctional complexes. Together, our results call for a reassessment of the structure and function of the submembrane cytoskeleton.

Highlights

Devoid of organelles and other cytoskeletal components, the human erythrocyte relies heavily on its membrane cytoskeleton to maintain structural stability and regulate membrane proteins
Estimates based on the copy numbers of spectrin molecules and the total area of the erythrocyte membrane have suggested the edges of the meshwork (d in Figure 1B) to be 70–80 nm (Lux, 2016; Vertessy and Steck, 1989; Waugh, 1982), close to the root-meansquare end-to-end distance of relaxed spectrin tetramers predicted from the experimental viscosity data of spectrin dimers (Stokke et al, 1985)
We started with human erythrocytes that were first chemically fixed and immunolabeled for super-resolution microscopy (Figures 1C–1E)

Summary

Introduction

Devoid of organelles and other cytoskeletal components, the human erythrocyte relies heavily on its membrane cytoskeleton to maintain structural stability and regulate membrane proteins. Current models often depict the erythrocyte cytoskeleton as a two-dimensional triangular meshwork (Figure 1A) composed of rod-shaped spectrin tetramers that connect at junctional complexes consisting of short actin filaments, adducin, tropomodulin, protein 4.1, and associated proteins (Alberts et al, 2015; Baines, 2010; Bennett and Gilligan, 1993; Lux, 2016). Electron microscopy (EM) data of spread erythrocyte cytoskeletons show 200-nm meshwork edges (Byers and Branton, 1985; Liu et al, 1987; McGough and Josephs, 1990), consistent with the extended full length of spectrin tetramers (Shotton et al, 1979). A recent study using cryo-electron tomography of the membrane-removed cytoskeleton of mouse erythrocytes indicated a 46-nm edge length (Nans et al, 2011)

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Discussion

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