Comparison of incorporation of wild type and mutated actins into sarcomeres in skeletal muscle cells: A fluorescence recovery after photobleaching study

The α‐actin mutation G15R in the nucleotide‐binding pocket of skeletal muscle, causes severe actin myopathy in human skeletal muscles. Expressed in cultured embryonic quail skeletal myotubes, YFP‐G15R‐α‐actin incorporates in sarcomeres in a pattern indistinguishable from wildtype YFP‐α‐actin. However, patches of YFP‐G15R‐α‐actin form, resembling those in patients. Analyses with FRAP of incorporation of YFP‐G15R‐α‐actin showed major differences between fast‐exchanging plus ends of overlapping actin filaments in Z‐bands, versus slow exchanging ends of overlapping thin filaments in the middle of sarcomeres. Wildtype skeletal muscle YFP‐α‐actin shows a faster rate of incorporation at plus ends of F‐actin than at their minus ends. Incorporation of YFP‐G15R‐α‐actin molecules is reduced at plus ends, increased at minus ends. The same relationship of wildtype YFP‐α‐actin incorporation is seen in myofibrils treated with cytochalasin‐D: decreased dynamics at plus ends, increased dynamics at minus ends, and F‐actin aggregates. Speculation: imbalance of normal polarized assembly of F‐actin creates excess monomers that form F‐actin aggregates. Two other severe skeletal muscle YFP‐α‐actin mutations (H40Y and V163L) not in the nucleotide pocket do not affect actin dynamics, and lack F‐actin aggregates. These results indicate that normal α‐actin plus and minus end dynamics are needed to maintain actin filament stability, and avoid F‐actin patches.