Mechanism of incorporation of newly synthesized myosin alkali light chain into myofibril

The present work was designed to study the incorporation of newly synthesized myosin alkali light chain [MLC] molecules into myofibris. cDNA of fast skeletal muscle type of MLC tagged with green fluorescence protein [LC3f-GFP] was transfected into cultured chicken cardiomyocytes, and the assembly of expressed LC3f-GFP was observed in living cells under a fluorescence microscope equipped with a cooled CCD camera. At 14-16 hours after transfection, LC3f-GFP was diffusely distributed in the cytoplasm of cardiomyocytes. In some cells, however, intense fluorescence spots of LC3f-GFP were found along myofibrils with a periodically of 1.2 micro m. Confocal microscopy of such cells, stained with rhodamine-labeled phalloidin, revealed the fluorescence spots of LC3f-GFP localized at both ends of A-bond. When these cells were further incubated, LC3f-GFP came to be localized at all levels of the A-bands by 26 hours after transfection. These results indicate that myosin filaments are not replaced with newly synthesized myosin molecules at once along their length, but molecules in filaments are replaced individually from their ends