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Activation of G-protein-coupled chemoattractant receptors triggers dissociation of Galpha and Gbetagamma subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PH(Crac)-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When naive cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PH(Crac)-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PH(Crac)-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity.

molecular biology of the cell

Quantitative Imaging of Single Live Cells Reveals Spatiotemporal Dynamics of Multistep Signaling Events of Chemoattractant Gradient Sensing in<i>Dictyostelium</i>

Quantitative Imaging of Single Live Cells Reveals Spatiotemporal Dynamics of Multistep Signaling Events of Chemoattractant Gradient Sensing inDictyostelium