Fig. 6. Live in vivo imaging of BRP at remodeling AZs.

(A to N) Live sptPALM imaging of endogenously tagged BRPmEOS3.2 line containing a mEOS3.2 tag at the stable MiMIC insertion site (brp[MI02987]), visualized at muscle 4 or 6/7 NMJs, performed in 1.5 mM Ca²⁺ 5 min after a 10-min incubation in HL3 with PhTx or in plain HL3 (Con) and imaging at 30 min after PhTx treatment. Images show representative PALM localizations (A and B), trajectory maps (C and D), and the application of tessellation analysis (E to H). Scale bars, 1 μm (B to G) and 200 nm (H and I). (J and K) Quantification of diffusion coefficients and radius of confinement from live BRPmEOS3.2 sptPALM imaging. Control NMJs: N = 7 images; 1443 trajectories from three animals; PhTx-treated NMJs: N = 9 images; 1761 trajectories from three animals. (K to N) Tessellation analysis from the same sptPALM dataset analyzed the diameters (K and M) and densities (L and N) of BRPmEOS3.2 localizations within AZ (K and L) and NC cluster (M and N) boundaries. Control: N = 26 ROIs, 406 AZs (Con) from 11 animals; PhTx: N = 26 ROIs, 226 AZs from 7 animals included for either condition. n = 2 sets with concurrent controls. Statistical significance is denoted as asterisks: ****P < 0.0001. Data distribution was statistically tested with a Kolmogorov-Smirnov test.