Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range of Dictyostelium cells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis.eukaryotic chemotaxis | gradient sensing | dynamic range extension | heterotrimeric G protein C hemotaxis in eukaryotic cells is observed in many physiological processes including embryogenesis, neuronal wiring, wound healing, and immune responses (1, 2). Chemotactic cells share basic properties including high sensitivity to shallow gradients and responsiveness to a wide dynamic range of chemoattractants (3, 4). For instance, human neutrophils and Dictyostelium cells can sense spatial differences in chemoattractant concentration across the cell body in shallow gradients as low as 2% and exhibit chemotaxis over a 10 5 -10 6 -fold range of background concentrations (5-7). Thus, wide-range sensing and adaptation are critical features of chemotaxis as well as other sensory systems such as visual signal transduction (8). However, the underlying regulatory mechanisms in eukaryotic chemotaxis remain unclear.The molecular mechanisms of chemotaxis are evolutionarily conserved among many eukaryotes that use G protein-coupled receptors (GPCRs) and heterotrimeric G proteins to detect chemoattractant gradients (3, 4). In Dictyostelium cells, extracellular cAMP works as a chemoattractant, and binding to its receptor cyclic AMP receptor 1 (cAR1) activates G proteins (Gα2Gβγ) along the concentration gradient, leading to the activation of multiple signaling cascades including the PI3K-PTEN, TorC2-PDK-PKB, phospholipase A2, and guanylyl cyclase pathways. In contrast to the spatial distributions of cAMP/cAR1 association and G-protein activation, downstream signaling pathways are activated in an extremely biased manner at the anterior or posterior of the cell (3, 4). For example, localized patches of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) are generated at the plasma membrane by an intracellular signal transduction excitable network (STEN) and function as a cue to control the pseudopod formation of...

In this chapter, we describe methods to monitor signaling events at the single-molecule level on the membrane of living cells by using total internal reflection fluorescence microscopy (TIRFM). The techniques provide a powerful tool for elucidating the stochastic properties of signaling molecules involved in chemotaxis of the cellular slime mold Dictyostelium discoideum. Taking cAMP receptor 1 (cAR1) as an example of a target protein for single-molecule imaging, we describe the experimental setup of TIRFM, a method for labeling cAR1 with a fluorescent dye, and a method for investigating the receptor's lateral mobility. We discuss how the developmental progression of cells modulates both cAR1 behavior and the phenotypic variability in cAR1 mobility for different cell populations.

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass.