Figure 10. Syt-1 and Syt-7 are found on the outside of Chromogranin-A-positive vesicles.

(A, E) Single optical slices of WT mouse chromaffin cells stained against CgA (α-CgA) and Syt-7 (α-Syt-7) or Syt-1 (α-Syt-1) acquired with 3D-structured illumination microscopy (3D-SIM). Scale bar: 5 µm. Bottom right: Magnified ROIs. ROI 1: a region of the cell from the merged-channel image. Scale bar: 2 µm. ROI 2: An example of a CgA-vesicles with Syt-7 or Syt-1 spots localized adjacent to it (top panels: α-CgA; middle panels: α-Syt-7 or α-Syt-1; bottom panels: merged image of CgA and Syt-7 or Syt-1 channels). Scale bar: 0.2 µm. (B, F) Quantification of spot numbers (mean ± SEM) for Syt-1, Syt-7 and CgA in WT and Syt-7 KO cells using the DiAna plugin for ImageJ. The number of spots was quantified in a 3D-volume with z-length of 2.1 µm around the middle of the cell (note that the numbers in Figure 9 are for the entire cell and estimated using another plugin). (C, G) Volumes of Syt-1, Syt-7 and CgA spots (mean ± SEM) calculated using the DiAna plugin. (D, H) Center-to-center distances between segmented spots from the CgA-channel (CgA) and their closest neighbor in the Syt-7 or Syt-1 channel. The histogram of distance distribution (black) shows that the center of the nearest Syt-7 and Syt-1 spot is localized at 0.22 and 0.19 µm from the center of CgA vesicles, respectively (mean of Gaussian fits). The histogram of partially randomized distance distributions (orange) obtained after inversion of the CgA-stack shows that the center of the nearest Syt-7 and Syt-1 spot are localized at a larger distance (0.32 and 0.37 µm, respectively, mean of Gaussian fits). A gray dashed line identifies the minimal distance center-to-center between two spots localized side-by-side, calculated by adding the mean radii of CgA- and Syt-spots. A pink dashed line represents the cumulative distribution of the center-to-center distances between spots from the two images. A purple dashed line represents the cumulative distribution of the randomized distances between spots. Data information: Data are presented as mean ± SEM. ****p<0.0001 (Mann-Whitney test). Number of cells: CgA/Syt-7: N = 19 cells; CgA/Syt-1: N = 22 cells.

Figure 10—figure supplement 1. Proposed role of Syt-7 and ubMunc13-2 in dense-core vesicle priming.

Syt-7 recruits vesicles into a critical distance (6–10 nm), where ubMunc13-2 can bridge vesicle and plasma membrane and stimulate SNARE-complex formation. The ubMunc13-2-dependent priming-step is promoted by phorbol esters (PMA/DAG) and can be stabilized by blocking NSF-dependent de-priming. At high prestimulation Ca²⁺, other molecular calcium-sensors likely contribute to Ca²⁺-dependent recruitment of vesicles to the plasma membrane.