Post-translational incorporation of actin into myofibrils in vitro: evidence for isoform specificity

The incorporation of actin into myofibrils has been examined in a cell-free system [Bouché et al.: Journal of Cell Biology 107:587-596, 1988; Goldfine et al.: Cellular and Molecular Biology of Muscle Development, 1989]. Actin was translated in a reticulocyte lysate in the presence of 35S-methionine (35S-actin) or purified from muscle and labeled with fluorescein-5-isothiocyanate (FITC-actin). Myofibrils were incubated with either 35S-actin or FITC-actin and then analyzed by gel electrophoresis or fluorescence microscopy. When myofibrils were incubated with FITC-actin monomer in the reticulocyte lysate buffer, strong fluorescent labeling was observed in Z-band regions and less so in I-bands. No fluorescence was detected in non-overlap regions of A-bands. Confocal microscopic analysis of these myofibrils indicated that FITC-actin was distributed evenly across the diameter of the myofibrils. These observations suggest that actin incorporation in the reticulocyte lysate buffer occurred at sites in the sarcomere which contain actin. In contrast, FITC-actin showed a variety of non-physiological incorporation patterns when incubated with myofibrils in the presence of an isotonic buffer (I-buffer). However, when ATP was added to I-buffer, FITC-actin showed a pattern of incorporation into myofibrils similar to that seen in the reticulocyte lysate buffer. Immunoblots indicated that actin of native size was released from myofibrils during incubation in the reticulocyte lysate buffer. No actin release was detected when the myofibrils were incubated in I-buffer lacking ATP. We used this system to compare the incorporation of actin isoforms into myofibrils. Both alpha- and beta-actins exhibited incorporation into the myofibrils but there was a three-fold greater incorporation of the alpha isoform. We propose that the differential affinities of actin isoforms for myofibrils and other cytoskeletal structures could provide a mechanism for actin isoform targeting within the cytoplasm.