SUB-Immunogold-SEM reveals nanoscale distribution of submembranous epitopes

TL;DR

A new scanning electron microscopy technique called SUB-immunogold-SEM allows for precise detection of proteins near cell membranes at the nanoscale level.

Contribution

SUB-immunogold-SEM introduces a novel method for detecting intracellular protein epitopes near the membrane with high sensitivity and quantification.

Findings

SUB-immunogold-SEM successfully localized Myosin rings at the tip of auditory hair cell stereocilia.

The method mapped the distribution of ACE2 receptors along the motile cilia of respiratory multiciliate cells.

Four critical sample preparation factors were identified to enhance the method's sensitivity.

Abstract

Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identified four critical sample preparation factors that contribute to the method’s sensitivity and validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane proteins. We evaluated the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale Myosin rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM provides a novel solution for nanoscale protein localization at the exposed surface of any cell.

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