Overcoming efflux of fluorescent probes for actin imaging in living cells

Actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We addressed this issue by constructing a series of probes which employ modified rhodamine fluorophores. We found that the best performing probes are based on 6-carboxy-carbopyronine scaffold. These probes show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We demonstrate their excellent performance in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.