Determination of Biomolecular Oligomerization in the Live Cell Plasma Membrane via Single-Molecule Brightness and Co-localization Analysis

Abstract

Oligomerization of biomolecules on the plasma membrane drives vital cellular functions. Despite the importance of detecting and characterizing these molecular interactions, there is an apparent lack of proper techniques capable of unraveling the exact composition of multimolecular complexes directly on the (live) cell membrane, particularly when present at high surface densities. In this chapter, commonly applied single-molecule fluorescence microscopy approaches are reviewed in terms of their suitability for detecting molecular aggregates down to the smallest biologically relevant unit: a molecular dimer. Amongst these techniques, Thinning Out Clusters while Conserving Stoichiometry of Labeling (TOCCSL) – a modality based on single-molecule brightness and co-localization analysis – is discussed in detail. The chapter lists basic principles of TOCCSL, its implementation on single-molecule fluorescence microscopes, analysis of brightness and co-localization-based microscopy data, and guidelines on how to choose parameters and labels to determine the number of labeled subunits within a multimolecular assembly. At the end, a tabular list of TOCCSL applications conducted so far is provided.