Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.
Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10-15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested that myosin fluorescence was localized at the A-bands of myofibrils. In addition, close examination of the fluorescent myosin bands indicated that they were composed of two fluorescent bars separated by a nonfluorescent line that corresponded to the H-zone. Once incorporated, the myosin underwent a relatively slow exchange along myofibrils as indicated by fluorescence recovery after photobleaching. Glycerinated myofibrils were able to bind fluorescent myosin in a similar pattern in the presence or absence of MgATP, indicating that actin-myosin interactions had little effect on this process. Fluorescent heavy meromyosin did not incorporate into myofibrillar structures after injection. Light meromyosin, however, associated with A-bands as did whole myosin. These results suggest that microinjected myosin, even with its relatively low solubility under the cytoplasmic ionic condition, is capable of association with physiological structures in living muscle cells. Additionally, the light meromyosin portion of the molecule appears to be mainly responsible for the incorporation.
Johnson
C S
CS
Department of Molecular and Cellular Biology, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
McKenna
N M
NM
Wang
Y
Y
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002642:Chick Embryo; D019793:Fluorescein; D005452:Fluoresceins; D066298:In Vitro Techniques; D008845:Microinjections; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000648:ultrastructure*; D009210:Myofibrils / Q000648:ultrastructure; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000008:administration & dosage / Q000378:metabolism*
D005452:Fluoresceins; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D009218:Myosins; D019793:Fluorescein