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Single-molecule kinetic and dynamic analyses of the biochemical reactions in living cells are one of the most useful methods of investigating the molecular mechanisms of cellular reactions, especially those involved in signal transduction on the plasma membrane. Here, we focus on single-molecule analyses of the intracellular signaling from RAS to RAF, which occurs on the plasma membrane. RAS and RAF are cell signaling proteins that regulate various cellular behaviors, including cell fate determination for proliferation and differentiation. Using single-molecule techniques in living cells, including single-pair Förster resonance energy transfer (FRET) detection and single-molecule tracking, we directly measured the elementary processes in the reactions between RAS and RAF, including the dissociation of RAS and RAF, the conformational changes in RAF, and the diffusion of RAS and RAF along the plasma membrane. Based on the results of these measurements, we discuss how the mutual molecular recognition between RAS and RAF facilitates accurate signal transduction. [ABSTRACT FROM AUTHOR]

Signaling pathways mediated by receptor tyrosine kinases (RTKs) are among the most important pathways regulating various functions and behaviors in mammalian cells. Although many studies performed over several decades have revealed the molecular mechanisms underlying the cellular events regulated by these pathways, the overall structures of the pathways remain unclear, especially their quantitative properties. A technology has emerged that can potentially address these issues. Recent developments in optical microscopy and molecular biology allow us to visualize the behaviors of single RTK molecules and their association partners with fluorescent probes in living cells. Using the quantitative nature of these single-molecule measurements, we studied the signaling of epidermal growth factor (EGF) and nerve growth factor (NGF), both of which stimulate RTK systems. Single-molecule analyses revealed molecular dynamics and kinetics that cannot be demonstrated with conventional biochemical methods. These include the kinetic transitions of these receptors induced by ligand binding, signal amplification by the dynamic interactions between active and inactive receptors, downstream signaling with a memory effect exerted by the receptor molecule, and shifts in the motional modes of ligand-receptor complexes. These novel insights obtained from single-molecule studies suggest that detailed models of RTK signaling, which involve signal processing depend on protein dynamics. [ABSTRACT FROM AUTHOR]