Abstract
Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.
Highlights
Enhanced Green fluorescent protein (GFP) from Aequorea victoria and its other derivatives including cyan and yellow fluorescent proteins (CFP and YFP, respectively) are widely used due to their narrow emission spectra, photostability, and low cellular toxicity [1]
As a first step to characterize an approach for 3- and 4-color live cell imaging in yeast, we generated C-terminal tagging cassettes for monomeric teal fluorescent protein 1 (mTFP1) and other FPs and tagged Cit1p (Citrate Synthase 1), an abundant citric acid cycle protein that localizes to the mitochondrial matrix [14], at its C-terminus with mTFP1, GFP, mCitrine, or mCherry
We find that Cit1p that is fused to each of these FPs localizes to structures that resemble mitochondria, i.e. long tubular structures that align along the mother-bud axis and accumulate in the bud tip
Summary
Enhanced Green fluorescent protein (GFP) from Aequorea victoria and its other derivatives including cyan and yellow fluorescent proteins (CFP and YFP, respectively) are widely used due to their narrow emission spectra, photostability, and low cellular toxicity [1]. Expression of FP-fusions by tagging well-characterized proteins of interest at their chromosomal locus provides a means to test whether the tag perturbs function, express tagged proteins at endogenous levels and obtain more uniform FP signal within a cell population. We characterized tagging cassettes for insertion of FPs into the yeast genome, demonstrated that the tags can be used for 3- and 4-color imaging in living cells, and describe the benefits of multicolor imaging with these cassettes. (BFP)/GFP/RFP [2] or CFP/YFP/RFP [3] are used for three-color live-cell in the yeast system. The UV illumination used for imaging of BFP in PLOS ONE | DOI:10.1371/journal.pone.0146120 January 4, 2016
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