Abstract
Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.
Highlights
Multicolour imaging of protein distributions in living cells is an important tool in the life sciences
In this study we developed a fast and reliable labelling protocol based on electroporation of living yeast cells expressing SNAP, CLIP, or Halo-tagged fusion proteins for dual colour live cell microscopy as well as for super-resolution STimulated Emission Depletion (STED) microscopy
We found that budding yeast cells expressing one of these fusion proteins could be readily labelled with TMR coupled to the appropriate substrate when the cell was chemically fixed and the cell wall was removed by treatment with zymolyase (Figure 1A)
Summary
Multicolour imaging of protein distributions in living cells is an important tool in the life sciences. Widely used self-labelling strategies are the SNAP-tag [3], the CLIP-tag [4], and the Halotag [5] methods. These labelling approaches rely on a selflabelling protein (the ′tag′), which can be genetically fused to a host protein. The self-labelling protein reacts covalently with an exogenously supplied substrate that is linked to a fluorescent dye. Because labelling by these strategies is irreversible and quantitative, they open up powerful options for live cell imaging approaches with a rich choice of different fluorophores
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
