Tomograms supporting data in figures 3-6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

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). 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.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.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.