Single-molecule localization microscopy reveals the molecular organization of endogenous membrane receptors

TL;DR

This paper introduces a new method using super-resolution microscopy to study the organization of membrane receptors in cells, which could improve personalized immunotherapy.

Contribution

The study introduces an optimized protocol for immunolabeling and analysis to accurately quantify and identify membrane receptor stoichiometry.

Findings

The method successfully resolved the molecular organization of CD45, CD69, and CD38 on Jurkat T cells.

The approach was applied to study interactions between multiple myeloma cells and therapeutic antibodies.

The protocol could enhance diagnostics and treatment using therapeutic antibodies.

Abstract

Super-resolution microscopy in combination with genetic labeling methods allows imaging of single proteins in cells. However, visualizing endogenous proteins on primary cells remains challenging due to the use of sterically demanding antibodies for labeling. Here, we demonstrate how immunolabeling conditions and antibody cross-linking influence the quantification and identification of membrane receptor stoichiometry on cells using single-molecule localization microscopy. We developed an optimized immunolabeling and analysis protocol and demonstrate the performance of the approach by resolving the molecular organization of endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the usefulness of the method for immunotherapy applications, we investigated the interaction of primary multiple myeloma cells with the therapeutic monoclonal antibodies daratumumab and isatuximab and a polyclonal anti-CD38 antibody. Our approach might lay the foundation for improved personalized diagnostics and treatment with therapeutic antibodies.

Single-molecule localization microcopy quantifies the expression and resolves the stoichiometry of endogenous membrane receptors.

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