Journal: eLife
Article Title: Endosomal chemokine receptor signalosomes regulate central mechanisms underlying cell migration
doi: 10.7554/eLife.99373
Figure Lengend Snippet: ( A ) Schematic representation of the experimental design used to measure luminescence upon proximity between Rap1GAP-SmBiT and LgBiT-FYVE in response to CCR7 activation. ( B ) Change in luminescence measured upon stimulation of HEK293-CCR7 cells expressing Rap1GAP-SmBiT and LgBiT-FYVE in response to 100 nM CCL19 or 100 nM CCL21 stimulation. The response to chemokine stimulation was normalized to vehicle control. ( C ) Area under the curve (AUC) was used to calculate the total response for each chemokine ligand. Data represent the mean ± SE of N=4 experiments. ( D ) Schematic representation of the enhanced bystander bioluminescence resonance energy transfer (EbBRET)-based assay to monitor proximity between RlucII-miniGi and the endosomal marker rGFP-Rab5 upon CCR7 activation from endosomes. ( E ) EbBRET measurements from CCR7-expressing HEK293 cells co-transfected with RlucII-miniGi and rGFP-Rab5 upon stimulation with 100 nM CCL19, 100 nM CCL21, or vehicle control. Data represents the mean ± SE from N=5 independent experiments. ( F ) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells co-transfected with the plasma membrane marker RFP-Lck and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 10 min. Representative images from N=3 experiments. ( G ) Co-localization quantification analysis of RFP-Lck and Halo-miniGi from six representative confocal microscopy images per condition. ( H ) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells co-transfected with the endosomal marker RFP-EEA1 and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 30 min. Representative images from N=3 experiments. ( I ) Co-localization quantification analysis of RFP-EEA1 and Halo-miniGi from five confocal microscopy images per condition. ( C, E, G, and I ) One-way ANOVA with Tukey’s multiple comparison post hoc tests were applied to determine statistical differences between the distinct treatments (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
Article Snippet: RFP-EEA1 (TagRFP-T-EEA1 cloned into pEGFP-C1 vector) and HA-Dyn-K44A (cloned into pcDNA3.1) constructs were gifts from Silvia Corvera and Sandra Schmid (respectively Addgene plasmids #42635 and #34683).
Techniques: Activation Assay, Expressing, Control, Bioluminescence Resonance Energy Transfer, Marker, Transfection, Confocal Microscopy, Imaging, Membrane, Comparison
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