RIα<sup>Y122A</sup> disrupts cAMP-stimulated RIα LLPS.
<p><b>(A)</b> Representative maximum intensity projections from confocal Z-stacks showing GFP expression <b>(left)</b> and quantification of puncta per cell <b>(right)</b> from HEK293T cells co-expressing Cα-mCherry plus GFP2-tagged RIα<sup>WT</sup&...
محفوظ في:
| المؤلف الرئيسي: | |
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| مؤلفون آخرون: | , , , , , , , , |
| منشور في: |
2025
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إضافة وسم
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| الملخص: | <p><b>(A)</b> Representative maximum intensity projections from confocal Z-stacks showing GFP expression <b>(left)</b> and quantification of puncta per cell <b>(right)</b> from HEK293T cells co-expressing Cα-mCherry plus GFP2-tagged RIα<sup>WT</sup> (WT, <i>n</i> = 90 cells) or RIα<sup>Y122A</sup> (Y122A, <i>n</i> = 59 cells) before (−) and after (+) Fsk and IBMX stimulation. ****P<i> </i>< 1 × 10<sup>−15</sup>; 2-way ANOVA with Šídák’s multiple comparisons test. <b>(B)</b> Representative fluorescence images and line-intensity profiles of the indicated regions in HEK293T cells expressing smAKAP-EGFP with mRuby2-tagged RIα<sup>WT</sup> or RIα<sup>Y122A</sup>. <b>(C)</b> BRET assay time-course <b>(left)</b> and quantification of Fsk-stimulated change <b>(right)</b> in the GFP2/RLuc8 emission ratio in HEK293T cells co-expressing GFP2-Cα plus RLuc8 fused to either RIα<sup>WT</sup> (<i>n</i> = 3 experiments) or RIα<sup>Y122A</sup> (<i>n</i> = 3 experiments). Untethered RLuc8 co-expressed with GFP2-Cα is used as a control. <i>P</i> = 0.61, unpaired, two-tailed Student <i>t</i> test. Solid lines indicate the mean, and shaded areas indicate SEM. <b>(D)</b> Critical saturation concentration (C-sat) curves of RIα<sup>Y122A</sup>. In vitro phase diagram of RIα<sup>Y122A</sup> vs. PEG 4000 <b>(Left)</b>. Filled circles represent droplets, and empty circles represent no droplets. C-sat curves showing PKA-Cα concentration-dependent LLPS behavior of RIα<sup>WT</sup> (green) and RIα<sup>Y122A</sup> (magenta) <b>(Right)</b>. Dotted lines indicate the boundary between one-phase and two-phase regions (shaded areas). RIα<sup>WT</sup> data were modified from our previous work [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3003262#pbio.3003262.ref010" target="_blank">10</a>]. <b>(E)</b> Quantification showing basal puncta number per cell in RIα-null MIN6 β cells expressing either mRuby2-tagged RIα<sup>WT</sup> (WT; <i>n</i> = 98 cells) or RIα<sup>Y122A</sup> (Y122A; <i>n</i> = 103 cells). ****<i>P</i> = 5.47 × 10<sup>−12</sup> (WT vs. Y122A); unpaired Komogorov-Smirnov test. Error bars in summary quantification indicate median ± 95% CI. <b>(F)</b> Representative images <b>(left)</b> and quantification of RIα<sup>Y122A</sup> puncta number (<i>n</i> = 53 cells) <b>(middle)</b> and area (for pre-existing puncta) (<i>n</i> = 15 puncta) <b>(right)</b> in RIα-null MIN6 β cells. ns, <i>P</i> = 0.17 for puncta number per cell; ns, <i>P</i> = 0.12 for pre-existing puncta area; paired, two-tailed Student’s <i>t</i> tests. All scale bars, 10 µm. The data underlying this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3003262#pbio.3003262.s010" target="_blank">S1 Data</a>.</p> <p>(TIF)</p> |
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