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

    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

    Design and topology of protein constructs.

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    (A-C) Legends for expected topology (A), protein length (B), and construct epitopes (C). (D) Protein topology schemes for the 15 constructs used. Below is a list with detailed information about the respective topology scheme of each construct depicted in (B). Uniprot accession numbers (acc.nr.) are available under https://www.uniprot.org/uniprot/. Sequences of all constructs are listed in “plasmid_sequence_information.xlsx” stored in “Plasmid_design.zip” available from http://dx.doi.org/10.17169/refubium-40101. No protein, used for background signals. (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

    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

    An analysis of the colocalization of epitope-tagged proteins to their expected compartments.

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    The images from S5 and S16 Figs were analyzed by measuring the Pearson’s correlation coefficient in different image regions. The box plot indicates the respective values, compared to a control, consisting of similar measurements across the same regions in the protein-of-interest channel, and mirrored regions in the compartment channel. All proteins show a colocalization that is significantly above the control values (Kruskal–Wallis test followed by Tukey post hoc test, p S1 Data file, Sheet “SFig 17_all_loc_func,” available from http://dx.doi.org/10.17169/refubium-40101. (TIF)</p

    Summary of the image data used for training and testing the deep network.

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    Number of confocal images obtained for each protein (with the given nanobarcode) as well as number of pixels that have been sampled for the deep learning dataset. All images have the same dimensions of 512 × 512 pixels. With 72-hour samples, each image contains 5 slices in a z-stack. Network training, validation, and testing are done only based on the subsampled pixels (with the numbers given in the last column), while the precision matrices in S20 Fig are obtained on full-frame images. (DOCX)</p

    Training and testing of the deep network.

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    (A) Pipeline through which data are prepared for training and testing the deep network for SNAP25 from 48-hour protocol as an example. Ten-dimensional vectors containing pixel-wise intensities across all channels are mapped along one dimension using kPCA transform. A relative threshold on the principal component separates foreground from background and results in a binary mask, based on which data can be gathered from points than contain proteins in the confocal image. (B) The result of Isomap, kPCA, t-SNE, and Sepctral Embedding “shallow-learning” methods for dimensionality reduction applied directly to the data gathered according to the pipeline explained in (A). (C) Training and validation accuracies averaged over all proteins in the dataset, sampled in each training epoch. Red dashed line shows the early stopping used based on the monitored validation accuracy. (D) Results of the ablation study, in which in each case one protein is removed from the training dataset and the performance of the deep network is evaluated based on the given metrics after training and validation procedure is performed. The data underlying this Figure are available as file “Fig 3_ABCD.xlsx” from http://dx.doi.org/10.17169/refubium-40101.</p
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