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

Abstract

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.

• Authors: Jushuo Wang, Yingli Fan, Balraj Mittal, Jean M Sanger, Joseph W Sanger
• Journal: Cytoskeleton (Hoboken, N.J.) (Cytoskeleton (Hoboken)) Vol. 79 Issue 9-11 Pg. 105-115 (09 2022) ISSN: 1949-3592 [Electronic] United States
• PMID: 36085566 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural)
• Copyright: © 2022 Wiley Periodicals LLC.

Chemical References

• Actins
• Nucleotides

Topics

• Humans
• Actins (genetics, metabolism)
• Sarcomeres (metabolism)
• Myofibrils (metabolism)
• Muscle, Skeletal (metabolism)
• Nucleotides (metabolism)