Multi-State Transition Kinetics of Intracellular Signaling Molecules by Single-Molecule Imaging Analysis.

The chemotactic signaling of eukaryotic cells is based on a chain of interactions between signaling molecules diffusing on the cell membrane and those shuttling between the membrane and cytoplasm. In this chapter, we describe methods to quantify lateral diffusion and reaction kinetics on the cell membrane. By the direct visualization and statistic analyses of molecular Brownian movement achieved by single-molecule imaging techniques, multiple states of membrane-bound molecules are successfully revealed with state transition kinetics. Using PTEN, a phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) 3'-phosphatase, in Dictyostelium discoideum undergoing chemotaxis as a model, each process of the analysis is described in detail. The identified multiple state kinetics provides an essential clue to elucidating the molecular mechanism of chemoattractant-induced dynamic redistribution of the signaling molecule asymmetrically on the cell membrane. Quantitative parameters for molecular reactions and diffusion complement a conventional view of the chemotactic signaling system, where changing a static network of molecules connected by causal relationships into a spatiotemporally dynamic one permits a mathematical description of stochastic migration of the cell along a shallow chemoattractant gradient.