Fig. 1: Adenylyl cyclases isoforms localize to primary cilia and endosomes.
From: Spatial organization of adenylyl cyclase and its impact on dopamine signaling in neurons
a–c Maximum intensity Z-projection of confocal microscopy images of MSNs expressing HA-AC3 (a), HA-AC5 (b), and HA-AC9 (c) and stained for cilia marker Arl13b. d Quantification of cilia localization by measuring the fraction of neurons with AC isoforms localized to cilia, using FLAG-D1R as cilia marker (not shown), and expressed as a percentage of total ciliated cells in the transfected cell population. Data are shown as mean ± s.e.m. from n = 3 independent experiments (total of 68–73 cells/condition). AC3 vs AC9, ***P = 0.0001, AC5 vs. AC9, ***P = 0.0002. e Cilia enrichment index calculated as a ratio of HA-AC fluorescence intensity in cilia (determined by Arl13b and FLAG-D1R) divided by total cell fluorescence. Data are shown as mean ± s.e.m. from n = 4 independent experiments (for AC3 and AC5) and n = 3 independent experiments (for AC9) (31–43 cells total/condition). AC3 vs. AC9, **P = 0.0083, AC3 vs. AC5, **P = 0.0085, AC5 vs. AC9, *P = 0.0175. f–h Representative confocal microscopy images of MSNs expressing AC3-GFP (f), AC5-GFP (g) and AC9-GFP (h) and stained for endosomal marker EEA1. Arrowheads indicate cilia. i Quantification of endosome localization by measuring the fraction of neurons with 10 or more AC-GFP internal puncta, expressed as a percentage of total transfected cells. Data are shown as mean ± s.e.m. from n = 4 independent experiments (34–47 cells total/condition). AC3 vs AC9, ***P < 0.0001, AC5 vs. AC9, ***P = 0.0002. j Endosome enrichment index calculated as a ratio of AC-GFP fluorescence intensity at EEA1-positive endosomes divided by total cell fluorescence. Data are shown as mean ± s.e.m. from n = 5 independent experiments; 38–58 cells total/condition. **P = 0.0012, *P = 0.0351. k Representative iSIM images from n = 3 independent experiments of MSN expressing AC9-GFP and stained for EEA1 and Vps35. Arrowheads indicate AC9-positive subdomains at the endosomal membrane. Scale bars are 10 μm. n.s. not significant, P values are calculated using unpaired two-tailed Student’s t-test. Source data are provided as a Source Data file.
