Exogenous gelsolin binds to sarcomeric thin filaments without severing.
We have investigated the binding of gelsolin to thin myofilaments in situ and their stability against severing. Differentiated myotubes from chicken skeletal muscle containing cross-striated myofibrils were permeabilized with Triton X-100 and incubated with gelsolin. Immunofluorescence microscopy localized both endogenous and exogenous gelsolin in the I-Z-I-regions of the sarcomers. The staining pattern suggested a binding of the exogenous gelsolin along the entire length of the thin filaments. This binding was Ca2+ dependent, but gelsolin was not removed after subsequent addition of EGTA. The fluorescence staining for actin remained unchanged after gelsolin incubation, indicating that thin filaments in cross-striated myofibrils were resistant to the severing action of gelsolin, in contrast to the microfilaments in stress fibers. After extraction of the permeabilized cells with high ionic strength to remove tropomyosin and myosin, gelsolin still bound along the entire thin filament and the actin pattern also remained unchanged. After Triton X-100 permeabilization and high ionic strength extraction, the giant protein nebulin was found to be still present as a myofibrillar component. Gelsolin treatment after high salt extraction affected neither actin nor nebulin in the thin filaments. We therefore conclude that nebulin confers the gelsolin resistance to the sarcomeric actin filaments.
Gonsior
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S
Biochemical Cell Biology Group, University of Bielefeld, Germany.
Hinssen
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H
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002642:Chick Embryo; D018260:Gelsolin / Q000378:metabolism*; D009124:Muscle Proteins / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012518:Sarcomeres / Q000378:metabolism*; D013552:Swine; D014335:Tropomyosin / Q000494:pharmacology
D018260:Gelsolin; D009124:Muscle Proteins; D014335:Tropomyosin; C035620:nebulin