Quantitative Imaging of Single Live Cells Reveals Spatiotemporal Dynamics of Multi-Step Signaling Events of Chemoattractant Gradient Sensing in Dictyostelium

Xuehua Xu,* Martin Meier-Schellersheim, ${dagger}$ Xuanmao Jiao,* Lauren E. Nelson,* and Tian Jin* ${ddagger}$

Laboratories of ^*Immunogenetics and Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852

Monitoring Editor: Paul Matsudaira

Activation of G-protein-coupledchemoattractant receptors triggers dissociation of G ${alpha}$ and G ${beta}$ ${gamma}$ subunits.These subunits induce intracellular responses that can be highlypolarized when a cell experiences a gradient of chemoattractant.Exactly how a cell achieves this amplified signal polarizationis still not well understood. Here, we quantitatively measuretemporal and spatial changes of receptor occupancy, G-proteinactivation by FRET imaging, and PIP₃ levels by monitoring thedynamics of PH_Crac-GFP translocation in single living cellsin response to different chemoattractant fields. Our resultsprovided the first direct evidence that G-proteins are activatedto different extents on the cell surface in response to asymmetricalstimulations. A stronger, uniformly applied stimulation triggersnot only a stronger G-protein activation but also a faster adaptationof downstream responses. When naïve cells (which have notexperienced chemoattractant) were abruptly exposed to stablecAMP gradients, G-proteins were persistently activated throughoutthe entire cell surface, whereas the response of PH_Crac-GFPtranslocation surprisingly consisted of two phases, an initialtransient and asymmetrical translocation around the cell membrane,followed by a second phase producing a highly polarized distributionof PH_Crac-GFP. We propose a revised model of gradient sensing,suggesting an important role for locally controlled componentsthat inhibit PI3Kinase activity.

Corresponding author. E-mail: tjin{at}niaid.nih.gov