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

Abstract

Summary: 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 25-60 nm. 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. : Pan et al. resolve the cytoskeletal organization in native erythrocytes using STORM super-resolution microscopy, revealing an ∼80-nm junction-to-junction distance that is in agreement with relaxed spectrin tetramers and showing that the junctional complexes are partially occupied by actin and its capping proteins. Keywords: erythrocyte, membrane cytoskeleton, spectrin-actin-based cytoskeleton, junctional complex, super-resolution microscopy, native ultrastructure, red blood cell