Chromophore-assisted laser inactivation of alpha- and gamma-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 specific 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 in 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.
Keppler
Antje
A
Virology, Hygiene Institute, University of Heidelberg, Heidelberg, Germany.
Ellenberg
Jan
J
D000818:Animals; D002460:Cell Line; D019793:Fluorescein / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry* / Q000502:physiology; D007834:Lasers; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis; D019853:O(6)-Methylguanine-DNA Methyltransferase; D017319:Photosensitizing Agents / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D026082:Singlet Oxygen / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
D005456:Fluorescent Dyes; D017319:Photosensitizing Agents; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D026082:Singlet Oxygen; D019853:O(6)-Methylguanine-DNA Methyltransferase; D019793:Fluorescein