Single-molecule fluorescence analysis of cellular nanomachinery components.

Abstract

Recent progress in proteomics suggests that the cell can be conceived as a large network of highly refined, nanomachine-like protein complexes. This working hypothesis calls for new methods capable of analyzing individual protein complexes in living cells and tissues at high speed. Here, we examine whether single-molecule fluorescence (SMF) analysis can satisfy that demand. First, recent technical progress in the visualization, localization, tracking, conformational analysis, and true resolution of individual protein complexes is highlighted. Second, results obtained by the SMF analysis of protein complexes are reviewed, focusing on the nuclear pore complex as an instructive example. We conclude that SMF methods provide powerful, indispensable tools for the structural and functional characterization of protein complexes. However, the transition from in vitro systems to living cells is in the initial stages. We discuss how current limitations in the nanoscopic analysis of living cells and tissues can be overcome to create a new paradigm, nanoscopic biomedicine.