29 research outputs found

    A functional assay to test nanobarcoded proteins.

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    Cells expressing different nanobarcoded proteins were pulsed with transferrin conjugated to Alexa488 and with EGF conjugated to Alexa647, for 10 minutes, allowing the cells to endocytose these ligands. Afterwards, they were immediately fixed or were chased (washed off) in a minimal buffer at 37°C, for 10 or 20 minutes. Finally, all cells were fixed and immunolabeled for the ALFA tag, to identify the nanobarcoded proteins. (A, B) The behavior of transferrin and EGF, respectively. Transferrin recycles, as expected, being released during the chase period (Kruskal–Wallis test followed by Tukey post hoc test, p N = 17–18 independent experiments. (C, D) Same data as above, but indicating the nature of the nanobarcoded protein in each of the independent experiments. The data underlying this Figure can be found in the following Sheets of the “S1 Data file: “Tf_SFig 6A,” “EGF_SFig 6B,” “Tf_SFig 6C,” and “EGF_SFig 6D.” The S1 Data file is available from http://dx.doi.org/10.17169/refubium-40101. (TIF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded Vti1a.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bars: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 7C_Vti1a,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (TIF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded syntaxin 13.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 14C_STX13,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (TIF)</p

    Endogenous SNAP25 and SNAP25(1100) have a similar cellular distribution within SNAP25(1100) transfected PC12 cells.

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    (A) Visualization of both endogenous SNAP25 and SNAP25(1100) using SNAP25 specific primary and secondary antibodies, plus NbALFA. (B, C) Negative control experiments, leaving out either primary antibodies (B) or NbALFA (C). (D) Imaging control, using a mixture of the same secondary antibody with 2 distinct fluorophores (targeting both endogenous SNAP25 and SNAP25(1100)), to provide a visual indication of the maximum expected colocalization. Bottom part of the figure: legend for used symbols and schemes. Scale bars: 2.5 μm. For quantification, see S17 Fig. (TIF)</p

    Neural network–based identification of nanobarcode proteins.

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    (A) Schematic of the neural network used for identification of nanobarcodes from pixel-wise fluorescence information. Brightness values across all emission channels are fed to the network as input, which, in turn, has been trained to predict the probability of this information pertaining to a specific nanobarcode, or a blank pixel. The trained network can readily be applied to full micrographs as well as stacks of images to produce false color outputs illustrating spatial distribution of proteins (further details in S18 Fig). (B) Example images of HEK293 cells transfected with specific nanobarcodes. To account for all possible emission features (including bleed-through), we acquired 11 frames for each area, consisting of the following: 405 nm excitation, with emission windows in blue, green, red, deep red; 488 nm excitation, with emission windows in green, red, deep red; 561 nm excitation, with emission windows in red and deep red; 633 nm excitation, with an emission window in deep red; brightfield. The panels in the left column show an overlay of the 4 brightest frames: 405 nm excitation, blue emission (in cyan); 488 nm excitation, green emission (in green); 561 nm excitation, red emission (in red); 633 nm excitation, deep red emission (in magenta). False color neural network output images are shown in the right column of (A). (C) Prediction accuracy of the neural network over a hold-out test dataset. For each protein, bars represent the precision (top), recall (middle), and F1-score (bottom). (D) False positive and false negative protein identifications (as percentage of all false predictions). For further details about the experimental procedures, imaging settings and neural network analysis, see the Methods section. For practical implementation purposes, we concentrated here on a subset of the labeled proteins, which were also used for the Nrxn/Nlgn experiments in Fig 4. Scale bars: 20 μm. The data underlying this Figure are available as file “Fig 2_CD.xlsx” from http://dx.doi.org/10.17169/refubium-40101.</p

    Transferrin and EGF imaging assays, tested for nanobarcoded LifeAct.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 12C_LifeAct,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (TIF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded VAMP4.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 15C_VAMP4,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (TIF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded syntaxin 4.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 8C_STX4,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (PNG)</p

    Visualization of 15 nanobarcode epitopes using 4 spectrally distinct nanobodies.

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    (A-D) Nanobody-based identification of the 4 genetically encoded nanobarcode epitopes mCherry(Y71L), GFP(Y66L), syn87, and syn2 and the ALFA-tag epitope by their corresponding nanobodies NbRFP, NbEGFP, NbSyn87, NbSyn2, and NbALFA. Scaling was optimized for each protein. (D) VAMP4(1111) example (all epitopes present) and negative control condition: mock transfection (no DNA, no epitopes present) using same intensity scale. (E) As in (D), now with upscale intensities. Scale bar: 50 μm. (TIFF)</p

    Deep network analysis results.

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    (A) The prediction accuracy matrix of trained deep networks, estimated over all the images in the dataset. To increase the complexity of the training and testing procedure, we expressed each construct for different time periods, and we then trained and tested the deep networks with all of these different datasets. Each row corresponds to a separate network that has been trained solely on the given dataset. Columns are the average pixel-wise prediction accuracy, assuming that all the pixels picked by the network in an image should belong to the protein with which the cells have been transfected. The given accuracy values may include effects of misexpressed proteins, weak fluorescence signals, and imaging noise. (B) From left to right, first column: merged channels (405 nm/CH1, 488 nm/CH2, 561 nm/CH3, 633 nm/CH4), before being processed by the network. Second column: images produced by assigning false colors to bright pixels, assuming that all the proteins in the image exactly match the given nanobarcode. Third column: output of the deep network, with each pixel given the false color representing the protein picked by the network. Colors are scaled based on class probabilities (Fig 2). Fourth column: false color output of the network overlaid on the gray “cell halos” produced from the brightfield images. Brightfield images have been processed to remove noise and background gradients and to enhance the contrast. (C, D) As (A) and (B), for additional nanobarcode proteins. The data underlying this Figure are available as file “FigS20_AC.xlsx” from http://dx.doi.org/10.17169/refubium-40101. (TIFF)</p
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