β- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequences; however, it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in β- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using β-actin 3'UTR; β-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single-molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in β- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.
Keywords: actin isoforms; cell biology; cell migration; coding sequence; mouse; translation rate.
Most mammalian cells make both β- and γ-actin, two proteins which shape the cell’s internal skeleton and its ability to migrate. The molecules share over 99% of their sequence, yet they play distinct roles. In fact, deleting the β-actin gene in mice causes death in the womb, while the animals can survive with comparatively milder issues without their γ-actin gene. How two similar proteins can have such different biological roles is a long-standing mystery. A closer look could hold some clues: β- and γ-actin may contain the same blocks (or amino acids), but the genetic sequences that encode these proteins differ by about 13%. This is because different units of genetic information – known as synonymous codons – can encode the same amino acid. These ‘silent substitutions’ have no effect on the sequence of the proteins, yet a cell reads synonymous codons (and therefore produces proteins) at different speeds. To find out the impact of silent substitutions, Vedula et al. swapped the codons for the two proteins, forcing mouse cells to produce β-actin using γ-actin codons, and vice versa. Cells with non-manipulated γ-actin and those with β-actin made using γ-actin codons could move much faster than cells with β-actin. This suggested that silent substitutions were indeed affecting the role of the protein. Vedula et al. found that cells read γ-codons – and therefore made γ-actin – much more slowly than β-codons: this also affected how quickly the protein could be dispatched where it was needed in the cell. Slower production meant that bundles of γ-actin were shorter, which allowed cells to move faster by providing a weaker anchoring system. Overall, this work provides new links between silent substitutions and protein behavior, a relatively new research area which is likely to shed light on other protein families.
© 2021, Vedula et al.