Labeling of Cytoskeletal Proteins in Living Cells Using Biotin Ligase Carrying a Fluorescent Protein

Abstract

Labeling with fluorescent proteins is now widely exploited for elucidating the functions and roles of target proteins in living cells. Previously, we developed a protein labeling method by combining a fluorescent protein with a biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). By taking advantage of this unique property, a target protein carrying BCCP in living cells can be labeled through biotinylation with BPL carrying a fluorescent protein. In the present work, to demonstrate the utility and performance of this labeling system in more detail, the cytoskeletal proteins β-actin and α-tubulin were selected as target proteins and labeled in living cells. With this approach, we succeeded in fluorescent imaging of actin filaments and microtubules in living cells, and shows the advantages of our approach over the conventional labeling methods with fluorescent proteins.