Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium.
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.
Xu
Xuehua
X
Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
Meier-Schellersheim
Martin
M
Jiao
Xuanmao
X
Nelson
Lauren E
LE
Jin
Tian
T
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D002630:Chemotactic Factors / Q000378:metabolism* / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000187:drug effects* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D018613:Microscopy, Confocal; D009930:Organic Chemicals; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000187:drug effects; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015398:Signal Transduction
D002630:Chemotactic Factors; D000242:Cyclic AMP; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins; D049452:Green Fluorescent Proteins; D009930:Organic Chemicals; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C417664:Alexa594