Seeing is believing, and fluorescently tagged proteins have ushered in a major revolution in cell biology. Instead of observing the static components of dead cells fixed in plastic and reacted with dyes, tagged proteins fluorescing a variety of colors can be tracked in real time in live cells and organisms. We can peek at the previously only imaginable perpetual dynamism of life at the molecular level. In addition to turning us into spell-bound voyeurs, well-defined fluorescent tags also give us a hand-hold to isolate the binding partners of proteins of interest.
In a recent article by the Rodal lab reported in Biology Open, the authors report a new tagging methods designed to get rid of technological artifacts that can cause fluorescently tagged proteins to be expressed at the wrong time and place, and at the wrong levels. By using CRISPR mediated gene editing in fruit flies, they developed a novel approach to visualize any protein of choice in any tissue of choice at the level, localization and time that nature has intended. This method, dubbed T-STEP (for tissue-specific tagging of endogenous proteins), opens up novel experimental avenues to answer long-standing questions in several areas of neuroscience and cell biology, such as: how many different neurotransmitters are expressed in one neuronal circuit? Which tissue-type is a protein expressed in and when? What happens to a disease carrying mutant protein in a tissue of interest at endogenous levels?
As a proof of principle, two endosomal proteins, Vps35 (linked to Parkinson’s disease) and OCRL (linked to Lowe syndrome), which have never before been seen or localized in fruit flies, have now been visualized live at endogenous levels. Moreover, a Parkinson’s disease-specific mutation (D620N) in Vps35 has also been tagged with fluorescent proteins, opening up exciting new research avenues for interrogating binding partners and/or kinetics that may be altered during the diseased states.
In summary, T-STEP is an exciting novel tool that offers a simple and efficient method to tissue-specifically tag any protein at endogenous levels. Authors from the Rodal lab include Kate Koles (Research Scientist) and Anna Yeh ’16.
- Koles K, Yeh AR, Rodal AA. Tissue-specific tagging of endogenous loci in Drosophila melanogaster. Biol Open. 2015;5(1):83-9.