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. 2015 May 15;10(5):1239-46.
doi: 10.1021/cb500957k. Epub 2015 Feb 16.

Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins

Cheryl A Telmer  1 , Richa Verma  1 , Haibing Teng  1 , Susan Andreko  1 , Leann Law  1 , Marcel P Bruchez  1
Affiliations

Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins

Cheryl A Telmer et al. ACS Chem Biol. .

Abstract

Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20-30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments.

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Conflict of interest statement

The authors declare the following competing financial interest(s): M.B. is a founder of Sharp Edge Labs, which aims to exploit FAP-based labeling for applications in drug discovery.

Figures

Figure 1
Figure 1
Diagram showing the cloning of targeting elements, FAPs and mCer3. All components were cloned as modules, except SKL was added into the reverse primer.
Figure 2
Figure 2
Confocal images showing the fluorescence pattern following expression of three different FAP-mCer3 tandems (side labels) targeted to five different cellular locations (top label). Signal from FAP_MGe (red) colocalizes with that of mCer3 (green), resulting in a yellow color in the merged images. Scale bars are 10 μm.
Figure 3
Figure 3
Flow cytometric analysis of stably expressing FAP-mCer3 HEK cells in different organelles and untransfected HEK cells. (A) Bar graph representation showing the median fluorescence intensity (MFI) of dL5**, dH6.2, and p13-CW labeled with 200 nM MGe in different organelles. For each organelle, the MFI of FAP_MGe was normalized to the maximum fluorescence signal across the various clones. (B) Expression level of mCerulean3 (represented in median fluorescence intensity) was analyzed from unlabeled FAP expressing HEK cells. The MFI for mCer3 expression was normalized to the maximum in each organelle across the various clones. Cells with no FAP-mCer3 expression (untransfected HEK cells) labeled with 200 nM MGe (A) or without (B) showed no fluorescence signal in either channel. Error bars are SD of independent duplicate measurements on stable selected cell populations.
Figure 4
Figure 4
No wash time course of fluorescence signal following addition of 200 nM MGe dye to HEK cells expressing dL5**-mCer3 in the cytosol, nucleus, mitochondria, peroxisomes, and ER. The mean fluorescence intensity for each time point for all the cells in the field of view was calculated, normalized to the maximum fluorescence, and plotted with the bands representing the SEM of multiple fields of view (n = 20 fields for cytosol, nucleus, peroxisomes, endoplasmic reticulum; n = 3 fields for mitochondria).
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