Journal: Molecular cell

Article Title: Molecular determinants and signaling effects of PKA RIα phase separation.

doi: 10.1016/j.molcel.2024.03.002

Figure Lengend Snippet: Figure 6. Non-canonical recruitment of PKA catalytic subunit to RIa condensates (A) Representative confocal images and line-in- tensity profiles of the indicated regions of HEK293T cells co-expressing RIa-GFP2 or RIaA99S-GFP2 plus Ca-mRuby2 (n = 10 cells from 4 independent experiments). Scale bars, 10 mm. (B) Pearson’s coefficient per RIa puncta in HEK293T cells co-expressing RIa-GFP2 (n = 2,288 puncta before and n = 2,502 puncta after stimulation) or RIaA99S-GFP2 (n = 1,055 puncta before and n = 1,253 after stimulation) with Ca-mRuby2. Effect size was calculated using Cohen’s d. Error bars indicate mean ± SD. (C) Schematic illustrating the classical model of dissociation of the canonical PKA holoenzyme (top) and the proposed model of the non-canonical R:C interaction (bottom). In the classical model, binding of cAMP (blue dots) to the CNB domains (open circles) causes complete dissociation of PKA-C (triangles), in contrast to the proposed non-canon- ical interaction in which active PKA-C remains tethered to RIa via the linker region. (D) Recovery kinetics (t1/2) of RIa puncta in HEK293T cells expressing RIa-GFP2 alone (RIa/; n = 35 puncta) or co-expressing Ca-mCherry plus either RIa-GFP2 (RIa/Ca; n = 51 puncta) or RIaA99S-GFP2 (A99S/Ca; n = 42 puncta) measured using FRAP. Error bars indicate median ± 95% CI. ****p = 6.15 3 106 (RIa vs. RIa/Ca), ***p = 0.000845 (RIa/Ca vs. A99S/Ca); ns, not significant; Brown-Forsythe and Welch ANOVA followed by Dunnett’s T3 multiple- comparisons test. (E) Average amplitude-weighted lifetimes measured from RIa puncta in HEK293T cells expressing RIa-GFP2 alone (RIa/; n = 39 cells) or co-express- ing Ca-mCherry plus either RIa-GFP2 (RIa/Ca; n = 54 cells) or RIaA99S-GFP2 (A99S/Ca; n = 50 cells). Error bars indicate mean ± SD. **p = 0.0066 (RIa/ vs. RIa/ Ca), ****p = 2.913 105 (RIa/Ca vs. A99S/Ca); ns, not significant; Brown-Forsythe and Welch ANOVA fol- lowed by Dunnett’s T3 multiple-comparisons test. (F) Normalized FluoSTEP-AKAR R/G emission ratio when RIa-FP11 (RIa; n = 41 cells), RIaA99S-FP11 (A99S; n = 42 cells), or RIa-FP11 and RIa(62–113)- mTagBFP (RIa/Linker; n = 38 cells) are expressed in RIa-KO HEK293T cells treated with 10 mM H89, followed by washout and addition of Fsk/IBMX. ****p = 1.98 3 105 (RIa vs. A99S), ****p = 6.95 3 105 (RIa vs. RIa/Linker); ns, not significant; un- paired, two-tailed Student’s t test. (G) Normalized AKAR4 Y/C emission ratio change in RIa-KO HEK293T cells treated with 10 mM H89, followed by washout and addition of Fsk/IBMX in the absence (KO; n = 69 cells) or presence of RIa-mRuby2 (RIa; n = 53 cells) or RIaA99S-mRuby2 (A99S; n = 87 cells) expression. Error bars indicate median ± 95% CI. ***p = 6.16 3 104 (KO vs. RIa), ****p = 1.23 3 105 (RIa vs. A99S); ns, not signifi- cant; Brown-Forsythe and Welch ANOVA followed by Dunnett’s T3 multiple-comparisons test. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pcDNA3 FluoSTEP-AKAR Zhang et al.21 N/A pcDNA3 AKAR4 Depry et al.55 N/A pcDNA3 Epac-SH188 Klarenbeek et al.79 Addgene plasmid #170349 Software and algorithms MATLAB (R2019a) MathWorks https://www.mathworks.com/ products/matlab.html PRISM (10.1.0) GraphPad https://www.graphpad.com/ scientific-software/prism/ Adobe Illustrator (26.5) Adobe https://www.adobe.com/ products/illustrator.html Fiji (ImageJ) NIH https://imagej.net/software/ fiji/downloads METAFLUOR (7.7) Molecular Devices https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy NIS-Elements AR (5.21.02) Nikon https://www.microscope.healthcare. nikon.com/products/software/ nis-elements/nis-elementsadvanced-research PyMOL Schrödinger, Inc. https://pymol.org/dokuwiki/ doku.php?id=installation Chimera UCSF https://www.cgl.ucsf.edu/chimera Coot Emsley et al.80 https://www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot/ MolProbity Williams et al.81 http://molprobity.biochem.duke.edu/

Techniques: Expressing, Binding Assay, Two Tailed Test