Functional fluorescence labeling of actins in live-cell system using engineered split-GFP technology

Actin filaments play essential roles in various cellular processes, and understanding their dynamics is crucial for studying cellular behaviors and actin-related diseases. However, conventional methods for visualizing actins often perturb its functionality or lack sufficient resolution for real-time imaging. In this study, we developed a method for functional fluorescence labeling of actin isoforms using split-GFP technology, specifically through insertion of a GFP11 tag into a flexible residue pair (T229/A230) of human β-actin (ACTB) or γ-actin (ACTG). This strategy (GFP11-i) was successfully applied to visualize actin dynamics in mammalian cell lines, including the effects of disease-related mutations (e.g., ACTB R196H and ACTG S155F). In addition, we demonstrated the labeling of actin filaments in Caenorhabditis elegans, further validating the cross-species applicability of this strategy. The GFP11-i methodology provides a versatile and powerful tool for investigating actin dynamics and cellular behaviors in both physiological and pathological contexts, facilitating the illustration of molecular mechanisms underlying actin-related diseases. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]