Actin Painting: a multicolor fluorescence staining method highlighting cell type-specific differences in the composition of filamentous actin architectures

Actin is a major structural component of the cytoskeleton in eukaryotic cells, and filamentous actin (F-actin) forms a variety of types of cellular structures. Many actin probes have been developed to visualize F-actin architectures in eukaryotic cells. However, it is known that double-stained images of F-actin obtained by two different types of actin probes often show partial inconsistencies. While developing actin probes, we observed that the merged images of three distinct actin probes, each labeled with a different fluorescent dye (red, green and blue), enabled various F-actin architectures to be distinguished based on color. This differentiation arises from slight variations in the distribution of each actin probe, and results in a unique color combination of actin probes that reflects the relative intensities of the three fluorescent dyes. In this report, we introduce a new cell staining method, named Actin Painting, which exploits the unique affinity of actin-binding molecules in each actin probe to F-actin architectures. This technique allows the classification of various cell types based on their specific actin cytoskeleton.