Figure 9.
The overall basal cytoskeletal link of AChRs is weakened in src-/-;fyn-/- myotubes but is still strengthened by agrin treatment. Wild-type (A) or src-/-;fyn-/- (B) myotubes were subjected to a first extraction in a buffer containing a low-detergent concentration, ranging from 0.03 to 0.09% Triton X-100 as indicated. Insoluble materials (pellets) were subjected to a second extraction, using 1% Triton X-100. AChRs were precipitated from the soluble low- and high-detergent fractions using α-BT and visualized by anti-AChR α subunit-antibodies in immunoblots. AChR extraction was quantified by densitometric scanning, and AChR signals are shown as a percentage of the sum of both extractions (low plus high detergent). Values are mean ± SEM from at least five experiments. For wild-type cells, 0.06% detergent extracts approximately one-half of the total AChRs, whereas for src-/-;fyn-/- myotubes, only 0.04% of detergent is required to achieve the same, indicating that, in the mutant, the AChR extractability is higher and thus the cytoskeletal link is weaker. C, Mutant cells were incubated overnight with agrin to induce AChR cluster formation. AChRs were first extracted with 0.05% and then with 1% detergent as described above and visualized by α-BT precipitation and AChR α immunoblotting. Agrin causes decreased AChR extractability, indicating stronger cytoskeletal linkage, because less AChRs are found in the first extraction and more in the second. D, Quantitation of experiments as in C shows a significant agrin-induced decrease of AChRs in the first extraction and a significant increase in the second extraction. Values are mean ± SEM from five experiments. *p = 0.03; unpaired Student's t tests. E, Sequential extraction as in C was performed for wild-type and C2C12 myotubes, using 0.06% Triton X-100 in the first extraction and 1% in the second. The percentage of decrease of AChRs in the first extraction, induced by agrin, was quantitated from five experiments and is the same, ∼30 - 40%, as for src-/-;fyn-/- myotubes. Thus, although the overall cytoskeletal link is weaker in src-/-;fyn-/- myotubes, the agrin-induced strengthening of this link is comparable with wild-type and C2C12 cells. Error bars represent SEM.