cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.25444/nhlbi.14519418
title: TIRF and PREM images supporting data in figure 3 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9) <br>It contains raw TIRF data for His-tag validation in fig. 3c,d. It also contains PREM images for fig 3e-g that show HeLa cells expressing Clathrin light chain A, Cavin1, and EPS15, respectively that were tagged with Ni-NTA-Au.<br>For TIRF correlation data, PC12 cells were were maintained in growth media containing DMEM, 10% fetal bovine serum, and 1% vol/vol penicillin/streptomycin. They were transiently transfected with His-GFP-Rab27a and mCherry-Rab27a to check for colocalization with DCVs. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. PC12 cells transfected with His-GFP-Rab27a were treated with Anti-Histidine-DyLight488 to test for accessibility of histidine epitope. TIRF imaging performed using an Olympus IX-81 inverted fluorescence microscope.<br>For PREM data, HeLa cells were transiently transfected with His-GFP-Clathrin Light Chain A, His-Cavin-GFP, and EPS15-GFP-His. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid, uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.
DOI: 10.25444/nhlbi.14519418.v1
title: TIRF and PREM images supporting data in figure 3 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9) <br>It contains raw TIRF data for His-tag validation in fig. 3c,d. It also contains PREM images for fig 3e-g that show HeLa cells expressing Clathrin light chain A, Cavin1, and EPS15, respectively that were tagged with Ni-NTA-Au.<br>For TIRF correlation data, PC12 cells were were maintained in growth media containing DMEM, 10% fetal bovine serum, and 1% vol/vol penicillin/streptomycin. They were transiently transfected with His-GFP-Rab27a and mCherry-Rab27a to check for colocalization with DCVs. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. PC12 cells transfected with His-GFP-Rab27a were treated with Anti-Histidine-DyLight488 to test for accessibility of histidine epitope. TIRF imaging performed using an Olympus IX-81 inverted fluorescence microscope.<br>For PREM data, HeLa cells were transiently transfected with His-GFP-Clathrin Light Chain A, His-Cavin-GFP, and EPS15-GFP-His. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid, uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.
DOI: 10.25444/nhlbi.14519436
title: PREM images supporting data in figure 4 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9) <br>It contains files for PC12 cells expressing His-tagged Rab3a, Rab27a, Rabphilin3A, Granuphilin-a and Rim2 and tagged with Ni-NTA-gold that are shown in fig. 4a-e.<br><br>HeLa cells were transiently transfected with His-tagged Rab3a, Rab27a, Rabphilin3A, Granuphilin-a and Rim2. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br><br>
DOI: 10.25444/nhlbi.14519511
title: PREM images supporting data in figure 6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9). <br>It contains EM images for PC12 cells expressing His-GFP-Syntaxin1A in fig 6a, and for His-GFP-SNAP25 in fig. 6d. These proteins expressing cells were treated with Ni-NTA-Au for gold labeling.<br>HeLa cells were transiently transfected with His-tagged Syntaxin1A and SNAP25. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br>
DOI: 10.25444/nhlbi.14519511.v1
title: PREM images supporting data in figure 6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9). <br>It contains EM images for PC12 cells expressing His-GFP-Syntaxin1A in fig 6a, and for His-GFP-SNAP25 in fig. 6d. These proteins expressing cells were treated with Ni-NTA-Au for gold labeling.<br>HeLa cells were transiently transfected with His-tagged Syntaxin1A and SNAP25. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br>
DOI: 10.25444/nhlbi.14519763
title: Tomograms supporting data in figures 3-6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9). <br>This file contains the tomograms used as representation in fig. 3h-j, fig. 4f-j, and 6d. It also contains all the underlying tomograms that were used in the generation of the gold particle distribution profiles in fig. 5c,d, and 6e,f. Fig 3h-j are for HeLa cells expressing His-Clathrin-light chain A, His-Cavin1-GFP, and EPS15-GFP-His. Fig. 4f-j are for PC12 cells expressing His-GFP-Rab3a, His-GFP-Rab27a, His-GFP-Rabphiliins3a, His-GFP-Granuphilin-a and His-GFP-Rim2. Fig. 6d is for PC12 cells expressing His-GFP-SNAP25. These cells were labeled with Ni-NTA-Au.<br>HeLa and PC12 cells were transiently transfected with His-tagged proteins of interest. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br><br>For distribution profiles, reconstructed tomograms were segmented using 3dmod, model editing and image display program. The 3dmod drawing tool was used to manually outline the optical sections of reconstructed vesicle tomograms with closed contours. This outline is a collection of coordinate points marking the locations of vesicle membrane in the image. Scattered points were used as separate objects to mark the independent gold particles. Coordinates from these two objects were used to obtain the normalized radius and height for each contour points from vesicle membranes and gold scattered points. <br>
DOI: 10.25444/nhlbi.14519763.v1
title: Tomograms supporting data in figures 3-6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9). <br>This file contains the tomograms used as representation in fig. 3h-j, fig. 4f-j, and 6d. It also contains all the underlying tomograms that were used in the generation of the gold particle distribution profiles in fig. 5c,d, and 6e,f. Fig 3h-j are for HeLa cells expressing His-Clathrin-light chain A, His-Cavin1-GFP, and EPS15-GFP-His. Fig. 4f-j are for PC12 cells expressing His-GFP-Rab3a, His-GFP-Rab27a, His-GFP-Rabphiliins3a, His-GFP-Granuphilin-a and His-GFP-Rim2. Fig. 6d is for PC12 cells expressing His-GFP-SNAP25. These cells were labeled with Ni-NTA-Au.<br>HeLa and PC12 cells were transiently transfected with His-tagged proteins of interest. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br><br>For distribution profiles, reconstructed tomograms were segmented using 3dmod, model editing and image display program. The 3dmod drawing tool was used to manually outline the optical sections of reconstructed vesicle tomograms with closed contours. This outline is a collection of coordinate points marking the locations of vesicle membrane in the image. Scattered points were used as separate objects to mark the independent gold particles. Coordinates from these two objects were used to obtain the normalized radius and height for each contour points from vesicle membranes and gold scattered points. <br>
DOI: 10.25444/nhlbi.14558388
title: Tomograms supporting data for His-GFP-Syntaxin1A in figure 6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9) <br>This file contains the tomograms used as representation in fig. 6a. It also contains all the underlying tomograms that were used in the generation of the gold particle distribution profiles in 6b,c. The cells in these tomograms were expressing His-GFP-Syntaxin1A and were labeled with Ni-NTA-Au.<br>PC12 cells were transiently transfected with His-tagged Syntaxin1A. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br>For distribution profiles, reconstructed tomograms were segmented using 3dmod, model editing and image display program. The 3dmod drawing tool was used to manually outline the optical sections of reconstructed vesicle tomograms with closed contours. This outline is a collection of coordinate points marking the locations of vesicle membrane in the image. Scattered points were used as separate objects to mark the independent gold particles. Coordinates from these two objects were used to obtain the normalized radius and height for each contour points from vesicle membranes and gold scattered points.
DOI: 10.25444/nhlbi.14558388.v1
title: Tomograms supporting data for His-GFP-Syntaxin1A in figure 6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

description: This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9) <br>This file contains the tomograms used as representation in fig. 6a. It also contains all the underlying tomograms that were used in the generation of the gold particle distribution profiles in 6b,c. The cells in these tomograms were expressing His-GFP-Syntaxin1A and were labeled with Ni-NTA-Au.<br>PC12 cells were transiently transfected with His-tagged Syntaxin1A. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.<br>For distribution profiles, reconstructed tomograms were segmented using 3dmod, model editing and image display program. The 3dmod drawing tool was used to manually outline the optical sections of reconstructed vesicle tomograms with closed contours. This outline is a collection of coordinate points marking the locations of vesicle membrane in the image. Scattered points were used as separate objects to mark the independent gold particles. Coordinates from these two objects were used to obtain the normalized radius and height for each contour points from vesicle membranes and gold scattered points.
DOI: 10.25444/nhlbi.17159351
title: Dual clathrin and integrin signaling systems regulate growth factor receptor activation

description: This collection comprises the platinum replica electron microscopy and light fluorescent data from the manuscript entitled:<br> "<b> </b>Dual clathrin and integrin signaling systems regulate growth factor receptor activation. " by Alfonzo-Mendez et al. 2022.<br> <br> Figure 1 of this manuscript compare the structure of clathrin in HSC3 cells genome edited to express endogenous levels of EGFR-GFP after stimulation with EGF (0, 2, 5, 15, 30, 60 min time points). All images are tiled montages acquired on an AMT XR111 ccd at 15000 x magnification (pixel size 1.2 nm). <b> </b> Figure 2-4 of this manuscript performs different drug treatments or siRNA to understand the impact on clathrin structure. Treatments include EGFR, Src and b5-integrin inhibitors (Gefitinib, PP2, and cilengitide, respectively) and siEGFR, siSRC and sib5-integrin. All images are tiled montages acquired on an AMT XR111 ccd at 15000 x magnification (pixel size 1.2 nm).<br> <br> Figure 5 of this manuscript shows the changes in correlation between clathrin and EGFR, Src or b5-integrin after EGF stimulation. Figure 6 of this manuscript shows the changes in correlation between clathrin and different b5-integrin mutants after EGF stimulation. Figure 7 of this manuscript shows the changes in correlation between clathrin and phosphorylated EGFR or Grb2 after EGF stimulation.<br><br>Software comprises MatLab codes used in the analysis of TIRF images to evaluate the correlation between clathrin and different proteins.<br>
DOI: 10.25444/nhlbi.17159351.v1
title: Dual clathrin and integrin signaling systems regulate growth factor receptor activation

description: This collection comprises the platinum replica electron microscopy and light fluorescent data from the manuscript entitled:<br> "<b> </b>Dual clathrin and integrin signaling systems regulate growth factor receptor activation. " by Alfonzo-Mendez et al. 2022.<br> <br> Figure 1 of this manuscript compare the structure of clathrin in HSC3 cells genome edited to express endogenous levels of EGFR-GFP after stimulation with EGF (0, 2, 5, 15, 30, 60 min time points). All images are tiled montages acquired on an AMT XR111 ccd at 15000 x magnification (pixel size 1.2 nm). <b> </b> Figure 2-4 of this manuscript performs different drug treatments or siRNA to understand the impact on clathrin structure. Treatments include EGFR, Src and b5-integrin inhibitors (Gefitinib, PP2, and cilengitide, respectively) and siEGFR, siSRC and sib5-integrin. All images are tiled montages acquired on an AMT XR111 ccd at 15000 x magnification (pixel size 1.2 nm).<br> <br> Figure 5 of this manuscript shows the changes in correlation between clathrin and EGFR, Src or b5-integrin after EGF stimulation. Figure 6 of this manuscript shows the changes in correlation between clathrin and different b5-integrin mutants after EGF stimulation. Figure 7 of this manuscript shows the changes in correlation between clathrin and phosphorylated EGFR or Grb2 after EGF stimulation.<br><br>Software comprises MatLab codes used in the analysis of TIRF images to evaluate the correlation between clathrin and different proteins.<br>