Figure 3.
Neuromuscular Junctions of Atg7−/− Myofibers Are Unstable and Fragmented
(A and B) Representative images of Atg7f/f and Atg7−/− neuromuscular junctions (NMJ) obtained using confocal in vivo microscopy. Muscles were in vivo pulse labeled with bungarotoxins (BGTs) conjugated with different fluorophores. BGT-Alexa Fluor 647 (shown in green) was injected 10 days before microscopy, thus identifying stable AChR, whereas BGT-Alexa Fluor 555 (shown in red), injected 1 hr prior to microscopy, identifies newly incorporated AChR. Micrographs show representative maximum z projections of confocal in vivo images of NMJs from Atg7f/f (A) and Atg7−/− (B). The panel on the right shows the quantification of AChR turnover as a function of NMJ fragmentation of adult and aged Atg7f/f versus Atg7−/−. Data are from at least four muscles of each group; ∗∗p < 0.01; ∗p < 0.05. A.U., arbitrary units. In the right panel of (B), values are mean ± SEM.
(C and D) Muscles were labeled with BGT-Alexa Fluor 555 48 hr before microscopy. In vivo confocal imaging was used to determine the amount of structures containing endocytic AChR. Micrographs show representative maximum z projections of confocal in vivo images of NMJs from Atg7f/f (C) and Atg7−/− (D). The panel on the right shows the average number of vesicular BGT-Alexa Fluor 555-positive structures per optical section. Data from 13 and 27 NMJs for Atg7f/f and Atg7−/−, respectively; ∗∗p < 0.01. In the right panel of (D), values are mean ± SEM.
(E) Immunohistochemistry of MuSK expression on aged Atg7f/f and Atg7−/− myofibers. Upper panel shows normal pattern of NMJs in Atg7f/f mice, where MuSK (red) localizes to NMJ, revealed by BGT-Alexa Fluor 647 (green). Lower panel shows MuSK (red) that accumulates inside myofibers at the level of NMJ (green). Panel on the right shows that the amount of diffused MuSK staining normalized over the total number of MuSK-positive NMJs is higher in aged Atg7−/− than Atg7f/f; ∗∗p < 0.01. In the right panel, values are mean ± SEM.