Abstract
Improved single-molecule methods can largely increase our understanding of underlying molecular mechanism during cellular signal transduction. In contrast to conventional bulk methods, monitoring molecules one at a time can circumvent averaging effects and acquire unique information. With single-molecule techniques, quantitative characterizations can be achieved at microscopic level, especially for biochemical systems with strong heterogeneity. Here we review four fundamental single-molecule techniques including total internal reflection fluorescence imaging, single-molecule fluorescence recovery after photobleaching, single-molecule Förster resonance energy transfer, and fluorescence correlation/cross-correlation spectroscopy. These techniques are frequently employed in quantitatively investigating the molecular translocation, protein-protein interactions, aggregations, and conformational dynamics involved in the signal transduction both in vitro and in vivo. We also summarized the basic principles and implementations of these single-molecule techniques, as well as the conjunct applications extending the single-molecule measurements to multiple dimensions.
Highlights
Signal transduction governing cellular activities is a complex system of communication
For the highly complex and dynamic signal transduction processes in living cells, real-time analysis on physiological and kinetic characteristics of biomolecules at single-molecule level can further our understanding of the regulation mechanisms of life activities [11, 12]
Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the diffusion and reaction properties of fluorescently labeled molecules based on bleaching and recovery of the fluorescent signals
Summary
Signal transduction governing cellular activities is a complex system of communication. With the advent of single-molecule detection (SMD) techniques [7,8,9], it is routine to image and track conformational changes, dynamics, and interactions of biomolecules at single-molecular level.
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