Phototoxicity and photoinactivation of blebbistatin in UV and visible light

Blebbistatin was recently identified as a selective, cell-permeant inhibitor of myosin II. Because blebbistatin is likely to be used extensively with fluorescence imaging in studies of cytoskeletal dynamics, its compatibility with common excitation wavelengths was examined. Illumination of blebbistatin-treated bovine aortic endothelial cells at 365 and 450-490 nm, but not 510-560 or 590-650 nm, caused dose-dependent cell death. Illumination of blebbistatin alone at 365 and 450-490 nm changed its absorption and emission spectra, but the resultant compounds were not toxic. In addition, photoreacted blebbistatin no longer disrupted myosin distribution in cells, indicating loss of pharmacological activity. Fluorescence microscopy showed that upon illumination, blebbistatin became bound to cells and to protein-coated glass, suggesting that toxicity may arise from light-induced reaction of blebbistatin with cell proteins. Blebbistatin should be used only with careful consideration of these photochemical effects.