Research ArticleRESEARCH METHODS

Versatile phenotype-activated cell sorting

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Science Advances  23 Oct 2020:
Vol. 6, no. 43, eabb7438
DOI: 10.1126/sciadv.abb7438
  • Fig. 1 Overall workflow and potential applications of the SPOTlight method.

    (Step 1) Cells of interest (e.g., bacteria, yeast, mammalian cells, or organoids) are made optically taggable by introducing a phototransformable FP or dye. These reagents can transform from a dim to bright state or from one color to another when excited with light of a specific wavelength. Some applications will screen naturally heterogeneous populations of cells, while others will screen libraries. siRNA, small interfering RNA. (Step 2) Cells are imaged for the phenotypes of interest. (Step 3) Phenotypes are quantified with single-cell resolution. Target cells are chosen based on the desired phenotypic profile. (Step 4) Target cells are optically tagged for retrieval by single-cell illumination. (Step 5) The tagged cells are detected and isolated using Fluorescence-activated cell sorting (FACS). (Step 6) The sorted cells are genotyped or further characterized as needed for the specific project. Additional rounds of screening can be performed if needed, for example, when conducting directed evolution experiments.

  • Fig. 2 Spatially-precise single-cell optical tagging and retrieval can be achieved with multiple cell types.

    (A and B) Individual bacteria, yeast, and human cells can be selectively photoactivated in crowded cultures, with minimal spurious photoactivation of neighboring cells. (A) Representative examples. Yellow arrows point to targeted cells. Scale bars, 20 μm. (B) Mean photoactivation fold change of target cells compared with their closest neighboring cells. The shaded regions represent the standard error of the mean (SEM). n = 7 (bacteria), 18 (yeast), and 5 (human) target cells. Equal numbers of neighboring cells were quantified. RFPo is the RFP intensity at t = 0. (C to E) SPOTlight enables the identification of photoactivated cells with high precision. (C) Schematics of the experimental strategy. (D) Human cells expressing EGFP and PAmCherry (GFP+ cells) were diluted 20-fold with cells expressing TagBFP and PAmCherry1 (BFP+ cells). In this representative experiment, 56 GFP+ cells were photoactivated. Out of 35 cells detected in the RFP+ sorting gate (left), 32 (~91%) were true positives while 3 were false positives (right). (E) Yeast cells expressing EGFP and PAmCherry1 were diluted 500-fold with cells expressing TagBFP and PAmCherry1. In this representative experiment, 96 GFP+ cells were photoactivated. Of 31 cells detected in the RFP+ sorting gate (left), 29 (~94%) were true positives while 2 were false positives (right). The photoactivated cell gates for (D) and (E) were determined using controls shown in fig. S4C. a.u., arbitrary units.

  • Fig. 3 A photoactivatable dye enables optical tagging and retrieval of human cells without genetically encoded transgenes.

    (A to C) Individual human cells stained with the photoactivatable dye PA-JF549 can be selectively tagged and retrieved. (A) Representative example of single-cell photoactivation. The yellow arrow points to the targeted cell. Merged images of the brightfield and red channels are shown. Scale bar, 20 μm. (B) Mean photoactivation fold change of target cells compared with their closest neighboring cells. F is the fluorescence; Fo is F at t = 0. The shaded regions represent the SEM. n = 12 cells per condition. (C) Photoactivated cells can be retrieved by FACS. In a representative example, 112 out of ~70,000 cells were individually photoactivated for 1 min. Seventy of the photoactivated cells (~63%) were recovered by FACS. (D to F) Whole enteroids stained with the photoactivatable dye PA-JF549 can be selectively tagged and their cells recovered by FACS. (D) Photoactivation of a representative enteroid. The red square shows the approximate area targeted for photoactivation. Merged images of the brightfield and red channels are shown. Scale bar, 50 μm. (E) Photoactivation fold change of target enteroids compared with their closest neighboring enteroids. The shaded regions represent the SEM. n = 11 enteroids per condition. (F) Cells from optically tagged whole enteroids can be recovered by FACS. Nine of 300 enteroids were each photoactivated for 1 min, pooled, and dissociated. A total of 241 individual cells were recovered by FACS.

  • Fig. 4 Automated photostability and brightness screening of ~ 3 million cells encoding mVenus variants.

    (A) Schematic of the experimental protocol. Photobleaching is performed with 508/25 nm light at 20 mW/mm2. (B) Expression cassette on the screening plasmid. (C to J) A representative round of screening. (C) Left: A representative field of view showing yeast cells expressing mVenus variants. Right: Magnified images and segmentation masks. Scale bars, 25 μm. (D) Photophysical properties of 287,186 individual cells from a representative library screening experiment. The color scheme represents the probability that each individual cell expresses a mutant with improved optical properties compared with its parental FP. The red circles indicate cells that were selected for optical tagging. (E and F) Representative example of three cells with different brightness (E) and photostability values (F). Cells are from the library depicted in (D). Scale bars, 1 μm. (G) A mosaic image of the 45 photoactivated cells before and after photoactivation. Scale bar, 10 μm. (H) RFP intensities of target cells before and after photoactivation. The black lines indicate the median values. (I) The photoactivation (RFP+) gate was designed using control samples with (left) and without (right) photoactivated cells. The ratio in blue indicates the number of cells inside the gate over the total number of cells analyzed. (J) Thirty of 45 optically tagged cells were detected and sorted by FACS.

  • Fig. 5 SPOTlight screening identified a YFP with up to fivefold improvement in photostability.

    (A and B) In cellulo characterization of mGold, the best YFP variant identified from screening. (A) mGold was more photostable than commonly used YFPs and exhibited similar brightness. Cells were photobleached with 508/25-nm light at 20 mW/mm2. YFP brightness was normalized for cell-to-cell differences in protein expression (YFP/BFP). The square markers indicate the means from six yeast cultures or six independent transfections. For each replicate, the mean photobleaching half-life and brightness of several hundred yeast or human cells were determined. The error bars represent the SEM. P ≤ 0.0001 for Welch’s analysis of variance (ANOVA) for both yeast and human cell data. ****P ≤ 0.0001 for Dunnett’s T3 post hoc test. (B) mGold is more photostable than mVenus over a range of irradiance levels. The mean photobleaching half-lives were computed from three yeast cultures or three independent transfections per irradiance level. For each replicate, the mean photobleaching half-life of several thousand yeast cells or hundreds of human cells was determined. The error bars represent the SEM. P = 0.013 (yeast) and ≤ 0.0001 (human cells) for t tests comparing the areas under the curve. **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 for t tests corrected for multiple comparisons with the Holm-Šídák method. (C) Time-lapse imaging experiment of representative HeLa cells expressing a fusion of keratin with mGold (top) or mVenus (bottom). Nuclei were identified by coexpressing a fusion of H2B and EBFP2. Cells were imaged every 30 s for 3 hours. Scale bars, 20 μm. (D) Expressing fusions of mGold and subcellular localization tags produced the expected pattern of fluorescence in HeLa cells. ER, endoplasmic reticulum. Scale bars, 10 μm.

  • Table 1 FPs used in this study.

    Note that Venus has incorrectly been used to refer to slightly different sequences in publications following the initial report of Venus (27, 28). The mVenus variant used in this work has been confirmed to be the monomeric (A206K) version of the original Venus by the first author of the publication reporting Venus (T. Nagai, pers. comm.) and has the same protein sequence (except A206K) as the Venus from the plasmid the Miyawaki group deposited on Addgene (#54859).

    Fluorescent proteinTypeSource
    TagBFPBFPGene synthesis using the sequence from Evrogen
    catalog no. FP171 as the template
    EGFPGFPpcDNA3.1-Puro-CAG-EGFP-P2A-mCherry used in Ref.
    (55), a generous gift from Michael Lin (Stanford
    University)
    CloverGFPpcDNA3.1-Puro-CAG-Clover-P2A-mCherry used in
    Ref. (54), a generous gift from Michael Lin (Stanford
    University)
    mClover3GFPGene synthesis using the sequence in Ref. (54) as the
    template
    sfGFPGFPAddgene #54579
    mNeonGreenGFPpcDNA3.1-Puro-CAG-mNeonGreen-P2A-mCherry
    used in Ref. (54), a generous gift from Michael Lin
    (Stanford University)
    EnvyGFPpcDNA3.1-Puro-CAG-Envy-P2A-mCherry used in Ref.
    (54), a generous gift from Michael Lin (Stanford
    University)
    mVenus*YFPpNCS-mVenus, generous gift from Michael Lin
    (Stanford University)
    mCitrineYFPAddgene #54723
    YPetYFPGene synthesis using the sequence in Ref. (57) as the
    template
    mCherryRFPpcDNA3.1-Puro-CAG-Clover-P2A-mCherry used in
    Ref. (54), a generous gift from Michael Lin (Stanford
    University)
    tdTomatoRFPAddgene #91767
    PAmCherry1PA-RFPAddgene #44855 for yeast experiments, and
    Addgene #31928 for bacteria and mammalian cell
    experiments
    PATagRFPPA-RFPAddgene #44854
    PAmKatePA-RFPAddgene #32692

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