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

Abstract

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.

Chemical tags: Applications in live cell fluorescence imaging

“…Chemical tags have been used to study the localization and dynamics of proteins in living cells, especially in experiments that can not be easily performed with fluorescent proteins. In the last few years, chemical tags have been used to label proteins with fluorophores suited for advanced imaging technologies such as super-resolution (SR) microscopy [57][58][59], Ca 2þ -imaging [60][61][62], pH sensing [63], hydrogen peroxide detection [64], chromophore assisted light inactivation [36,65,66], and multi-photon microscopy [19]. Recently, Kosaka et al demonstrated the use of the Halo-tag to perform in vivo imaging studies in live animals for the first time [67].…”

“…Keppler et al studied the effect of inactivation of a-and g-tubulin on cell division and spindle function [65]. Inactivation of fluorescein tagged SNAP-a-tubulin causes spindle shortening and arrest in metaphase, whereas inactivation of centrosomal g-tubulin affected microtubule nucleation and growth rate.…”

The chromosome peripheral proteins play an active role in chromosome dynamics

“…However, the side effect of RNAi cannot be excluded completely unless the proteome analysis is performed to confirm all protein expression level. To identify the roles of CPPs in the chromosome periphery, a temporal and spatial-specific knockdown method, for example, chromophore-assisted laser inactivation with a SNAP-tagged protein (84) or Killer-Red protein (85), would be effective.…”

Fluorescent Labeling of SNAP-Tagged Proteins in Cells

“…2 ). Examples of recent applications include single molecule [ 10 , 11 ] and super-resolution imaging [ 12 , 13 ], analysis of protein function [ 14 ], targeted protein inactivation [ 15 ], protein-protein interactions [ 16 , 17 ], protein-drug interactions [ 18 , 19 ], and the determination of protein half-life in animals [ 20 ]. Additionally, similar hAGTbased tag, named CLIP-tag, was developed recently [ 21 ].…”