Chromophore-Assisted Laser Inactivation of α- and γ-Tubulin SNAP-tag Fusion Proteins inside Living Cells

Chromophore-assisted laser inactivation (CALI) can help to unravel localized activities of target proteins at defined times and locations within living cells. Covalent SNAP-tag labeling of fusion proteins with fluorophores such as fluorescein is a fast and highly special tool to attach the photosensitizer to its target protein in vivo for selective inactivation of the fusion protein. Here, we demonstrate the effectiveness and specificity of SNAP-tag based CALI by acute inactivation of alpha-tubulin and gamma-tubulin SNAP-tag fusions during live imaging assays of cell division. Singlet oxygen is confirmed as the reactive oxygen species that leads to loss of fusion protein function. The major advantage of SNAP-tag CALI is the ease, reliability, and high flexibility labeling: the genetically encoded protein tag can be covalently labeled with various dyes matching the experimental requirements. This makes SNAP-tag CALI a very useful tool for rapid inactivation of tagged proteins in living cells.