Figure 1.
Induction of AChR endocytosis by crosslinking with biotin-BTX and QD-streptavidin (BBQ).
A, A schematic diagram and representative images showing 2 pools of AChRs revealed by BBQ labeling. The fluorescence images were captured at the focal plane in the middle of a muscle cell (depicted as a black dotted line in the schematic diagram). Arrows indicate internalized AChRs (in the red box); arrowheads indicate surface AChRs (in the green boxes). The colored, boxed regions were magnified by 5 times for clarity. B, Two representative fluorescence intensity profiles of BBQ signals of distinguishing internalized and surface AChRs. Internalized AChRs (red curve) showed higher fluorescence intensity compared with surface AChRs (green curve). Moreover, because single QDs blink, the fluorescence intensity (background-subtracted) of the surface AChRs occasionally dropped to zero. C, Two representative trajectories showing distinct movement behaviors of internalized and surface AChRs. Linear movement represents internalized AChRs, whereas Brownian motion represents surface AChRs. D, A representative image reconstructed from a z-stack maximal projection showing the subcellular localization of BBQ-labeled internalized AChRs. To outline the cell membrane (arrowheads), the ganglioside GM1 was labeled with biotin-CTX and then with streptavidin-QD (labeled as BCQ). E, Colocalization of BBQ-labeled internalized AChRs with the early endosomal marker EEA1 (arrow). Arrowheads indicate newly internalized AChRs prior to endosomal fusion. F, Correlation between AChR binding sites and internalized AChRs. The concentration of AChR binding sites was manipulated by either reducing the QD-streptavidin concentration (filled circles) or by masking the biotin-binding sites of QD-streptavidin with free biotin (open circles). Data are mean ± SEM; *p<0.01 (Student’s t test).
Figure 2.
BBQ crosslinking-induced AChR endocytosis through clathrin-dependent, caveolin-independent mechanisms.
A, Representative images showing the inhibition of crosslinking-induced AChR endocytosis by specific pharmacological inhibitors of clathrin (PAO and MDC) but not caveolin (MCD and filipin). For clarity, the boxed regions were magnified twofold, and the BBQ-labeled AChRs are marked with arrows in the insets. B, Quantification showing the effect of pharmacological agents on crosslinking-induced AChR endocytosis; n = 120, control; 90, vehicle, PAO, MDC, MCD, and filipin. Data are mean ± SEM; *p<0.001 (Student’s t test).
Figure 3.
Microtubules, but not actin filaments, are required for intracellular movement of endocytosed AChR vesicles.
A, Representative images showing the close association of BBQ crosslinking-induced AChR vesicles (BBQ) and microtubule structures (tubulin) in cultured muscle cells (arrows). The integrity of the microtubule network was effectively perturbed by treatment with nocodazole (Noc) or exposure to 4°C, which led to the fragmentation of the microtubule structures; however, microtubules were not affected by latrunculin A (Ltn A) treatment. Crosslinking-induced AChR endosomes were detected but were not associated with microtubule (arrowheads). The boxed regions were magnified by 8 times for clarity. B, Quantification showing the differential effects of cytoskeletal disruption of microtubules and actin filaments on the movement of crosslinking-induced AChR vesicles. Data are mean ± SEM; n = 30; *p<0.001 (Student’s t test).
Figure 4.
Inhibition of crosslinking-induced AChR endocytosis by agrin-MuSK signaling.
A, Representative images showing the attenuation of AChR endocytosis by agrin-MuSK-rapsyn signaling. Muscle cells were either treated with agrin or distinct forms of GFP-MuSK or GFP-Rapsyn (full-length: FL; truncated form: TR) were overexpressed in muscle cells. For clarity, the boxed regions were magnified twofold and are shown in the insets. B, Quantitative analysis of the number of BBQ-labeled AChR vesicles formed in response to the activation of agrin signaling or the overexpression of its downstream molecules MuSK and rapsyn. Data are mean ± SEM; n = 172, control; 151, agrin; 97, GFP-MuSK FL; 101, GFP-MuSK TR; 65, GFP-Rapsyn FL; 98, GFP-Rapsyn TR; *p<0.001 (Student’s t test).
Figure 5.
Dispersal of AChRs in aneural clusters triggered by crosslinking-induced endocytosis.
A, Representative images showing the presence of spontaneously formed aneural AChR clusters at various time points in cells in which AChR endocytosis was induced by crosslinking. Cultured Xenopus muscle cells were labeled with BBQ at 0 h and the dispersal of aneural AChR clusters was followed over a course of 24 h. For clarity, aneural AChR clusters, where present, are indicated by boxes and are magnified in the insets. B, Quantification showing the time course of BBQ crosslinking-induced formation of AChR vesicles; n = 86. C, Quantification showing the number of aneural AChR clusters in relation to time in cultured muscle cells with (filled marks) or without (open marks) BBQ crosslinking. Data are mean ± SEM; n = 101, control; 95, with BBQ crosslinking; *p<0.001 (Student’s t test).
Figure 6.
Enhancement of crosslinking-induced dispersal of aneural AChR clusters by synaptogenic stimulation.
A, Representative images showing the effects of synaptogenic signals on the dispersal of aneural AChR clusters triggered by BBQ crosslinking. The aneural AChR clusters (arrows) were counted in cultured muscle cells in the presence or absence of BBQ crosslinking, in combination with agrin treatment (middle 2 columns) or HB-GAM bead stimulation (last 2 columns). B, Quantification showing the synergistic action of AChR crosslinking and synaptogenic signaling on the dispersal of aneural AChR clusters. Data are mean ± SEM; *p<0.01 (Student’s t test).