Figure 3. Nanometer organization of P2Y₁₁ with Ca_V1.2 and PKA_cat in human arterial myocytes.

Representative conventional TIRF images (top) and corresponding GSD reconstruction maps (bottom) from human arterial myocytes labeled for (A) P2Y₁₁ and Ca_V1.2 and (B) P2Y₁₁ and PKA_cat. Lower panels display enhanced magnifications of areas shown in yellow boxes (scale bar, 400 nm). (C) Histograms of the area of P2Y₁₁, Ca_V1.2 and PKA_cat clusters in isolated human arterial myocytes (1621 ± 29, 1209 ± 16 and 1322 ± 20 nm², respectively; Figure 3—source data 1). (D) Bar plot of cluster density for P2Y₁₁, Ca_V1.2 and PKA_cat (38 ± 2, 29 ± 4, and 25 ± 3 clusters/µm², respectively; Figure 3—source data 2). Enlarged merged image (left) and associated x-y fluorescence intensity profile (right) from area highlighted by the dotted lines of sites of close proximity between (E) P2Y₁₁ (red) and Ca_V1.2 (green) and (F) P2Y₁₁ (red) and PKA_cat (green) (scale bar, 200 nm). Histograms of the lowest intermolecular distance to P2Y₁₁ centroids for (G) Ca_V1.2 (n = 19,611 particles from 6 cells; Figure 3—source data 3) and (H) PKA_cat (n = 22,425 particles from 6 cells; Figure 3—source data 4) fluorescence particles. Data were fit with a sum of two Gaussian functions with depicted R² and centroids. (I) Bar plot of % overlap of P2Y₁₁ with Ca_V1.2 or PKA_cat for experimental (Ca_V1.2: n = 36 segments from 12 cells; PKA_cat: n = 22 segments from 11 cells) and randomized simulations images (Ca_V1.2: n = 6 segments from 6 cells; PKA_cat: n = 6 segments from 6 cells) (*p<0.05, unpaired t test with Welch’s correction; Figure 3—source data 5).

Figure 3—figure supplement 1. Validation for PKA_cat primary antibody.

Representative confocal images of PKA_cat-associated fluorescence (top) and differential interference contrast (DIC, bottom) in wt mouse arterial myocytes stained with an anti-PKA_cat antibody (- PKA_cat blocking peptide on left side; n = 7 cells) and an anti-PKA_cat antibody preabsorbed with a PKA_cat blocking peptide (+PKA_cat blocking peptide on right side; n = 8 cells).

Figure 3—figure supplement 2. Nanometer organization of P2Y₁₁, Ca_V1.2 and PKA_cat in murine arterial myocytes.

Representative TIRF images (top) and corresponding GSD reconstruction maps (bottom) from murine arterial myocytes labeled for (A) P2Y₁₁ and Ca_V1.2 and (B) P2Y₁₁ and PKA_cat. Lower panels show enhanced magnifications of areas highlighted in yellow boxes (scale bar, 400 nm). (C) Histograms of the area of P2Y₁₁, Ca_V1.2 and PKA_cat clusters in arterial myocytes (1803 ± 22, 2111 ± 68, and 1836 ± 28 nm², respectively; Figure 3—figure supplement 2—source data 1). (D) Bar plot of cluster density for P2Y₁₁, Ca_V1.2 and PKA_cat (37 ± 2, 24 ± 2, and 23 ± 3 clusters/µm², respectively; Figure 3—figure supplement 2—source data 2). Enlarged merged image (left) and associated x-y fluorescence intensity profile (right) from area highlighted by the dotted lines of sites of close proximity between (E) P2Y₁₁ (red) and Ca_V1.2 (green) and (F) P2Y₁₁ (red) and PKA_cat (green) (scale bar, 200 nm). Histograms of the lowest intermolecular distance to P2Y₁₁ centroids for (G) Ca_V1.2 (n = 23,722 particles from 6 cells; Figure 3—figure supplement 2—source data 3) and (H) PKA_cat (n = 19,219 particles from 5 cells; Figure 3—figure supplement 2—source data 4) fluorescence particles. Data were fit with a sum of two Gaussian functions with depicted R² and centroids.

Figure 3—figure supplement 3. Negative controls for GSD images in human and murine arterial myocytes, and experimental and randomized reconstruction maps.

Representative TIRF images (top) and corresponding GSD reconstruction maps (bottom) from freshly dissociated (A) human (n = 6 cells from four humans) and (B) mouse (n = 6 cells from six mice) arterial myocytes labeled with secondary antibodies only (no 1° Ab, Alexa 647 or no 1° Ab, Alexa 568). (C) Representative binarized sub-image area of experimental GSD super-resolution localization maps (top) and randomized simulation distribution images (bottom) for P2Y₁₁ with Ca_V1.2 (left panels; experimental, n = 38 sub-image area from 12 cells; randomized, n = 6 sub-image area from 6 cells) and P2Y₁₁ with PKA_cat (right panels; experimental, n = 24 sub-image area from 11 cells; randomized, n = 6 sub-image area from 6 cells) in arterial myocytes. A smoothing filter was applied to the P2Y₁₁, Ca_V1.2 and PKA_cat images for presentation. Note that randomization data were repeated six times from six different experimental GSD super-resolution localization maps. The overlap images for both experimental and simulated data were generated by multiplying the P2Y₁₁ image by the respective Ca_V1.2 or PKA_cat image, thus revealing overlapping objects.