Quantification of intracellular N-terminal \u03b2-actin arginylation

Abstract

Actin is a ubiquitous, essential, and highly abundant protein in all eukaryotic cells that performs key roles in contractility, adhesion, migration, and leading edge dynamics. The two non-muscle actins, β- and γ-, are ubiquitously present in every cell type and are nearly identical to each other at the amino acid level, but play distinct intracellular roles. The mechanisms regulating this distinction have been the focus of recent interest in the field. Work from our lab has previously shown that β-, but not γ-, actin undergoes N-terminal arginylation on Asp3. While functional evidence suggest that this arginylation may be important to actin’s function, progress in these studies so far has been hindered by difficulties in arginylated actin detection, precluding estimations of the abundance of arginylated actin in cells, and its occurrence in different tissues and cell types. The present study represents the first antibody-based quantification of the percentage of arginylated actin in migratory non-muscle cells under different physiological conditions, as well as in different cells and tissues. We find that while the steady-state level of arginylated actin is relatively low, it is consistently present in vivo, and is somewhat more prominent in migratory cells. Inhibition of N-terminal actin acetylation dramatically increases the intracellular actin arginylation level, suggesting that these two modifications may directly compete in vivo. These findings constitute an essential step in our understanding of actin regulation by arginylation, and in uncovering the dynamic interplay of actin’s N-terminal modifications in vivo.