Fig 1: Gold nanoprobe labeling of DCV proteins imaged with 2D and 3D PREM.2D PREM images of PC12 cells transfected with His-tagged a Rab3a, b Rab27a, c Rabphilin3a, d Granuphilin-a, and e Rim2. The upper panel shows a representative crop from a larger PREM image of a cell (Supplementary Fig. 12) and the lower panel shows enlarged examples of individual DCV structures labeled with Ni-NTA-Au. Scale bars are 200 and 100 nm, respectively. Tomogram slice (XY view, scale bar = 100 nm) of an individual DCV structure labeled with Ni-NTA-Au for f Rab3a, g Rab27a, h Rabphilin3a, i Granuphilin-a, and j Rim2, and the XZ (scale bar = 100 nm) and YZ (scale bar = 50 nm) views for the z slice denoted by cyan dashed lines. Two independent imaging experiments were performed for 2D- and 3D-EM.
Fig 2: Correlative dSTORM and platinum replica EM analysis of proteins on DCVs.Correlative images of PC12 cells expressing NPY-mNG and Alexa Fluor-647-GFP-nanobody labeled dark GFP fused proteins a Rab3a, b Rab27a, c Rabphilin3a, and d Granuphilin-a. e Correlative image for endogenous Rim2 labeled with anti-Rim2 antibody. Scale bars are 500 and 200 nm for left and right panels, respectively. Images were cropped from larger images shown in Supplementary Fig. 11. For analysis, ten-pixel-sized bins created from the center of the f EM image and applied to the respective g super-resolved fluorescent image to plot fluorescence profiles. Yellow circle indicates the outline of vesicle, and pink and blue lines are bins within and outside of the vesicle edge. h Averaged and normalized fluorescence intensity profiles for dGFP-Rab3a (n = 8 cells; 458 vesicles), dGFP-Rab27a (n = 7 cells; 545 vesicles), dGFP-Rabphilin3a (n = 8 cells; 644 vesicles), dGFP-Granuphilin-a (n = 10 cells; 275 vesicles), and anti-Rim2 (n = 5 cells; 129 vesicles) (Supplementary Table 1a). Overexpression CLEM data were collected from two (Rab3a, Granuphilin-a) and three (Rab27a, Rabphilin3a) independent experiments and immunolabeled CLEM data from one experiment. The mean fluorescence intensity is shown as a dark line (black = Rab3a; red = Rab27a; blue = Rabphilin3a; magenta = Granuphilin-a), and the standard error of the mean is shown in transparency. The fluorescence intensity profiles for endogenous proteins including Rab3a, Granuphilin-a, and Rim2 are presented in Supplementary Fig. 5.
Fig 3: Expression of Dmxl2, WDR7 (protein), RAB3A (protein), Otof, Sh3gl2, Synj1, Dnm1, and Rab3a in the organs of Corti of the Dmxl2 CKO and control mice at 1 month of age. A, FISH staining shows disrupted Dmxl2 mRNA expression in the CKO mice. Red and green: probes targeted to (KO) and outside of (un-ko) the deleted exons 2–4 region, respectively. Scale bars: 50 μm. The positions and the sequences of the FISH probes for Dmxl2 are provided in Extended Data Figure 6-1 and Table 6-2. B, Immunofluorescence staining shows decreased expression of WDR7 and normal expression of RAB3A at the protein level. Scale bars: 10 μm. C, Expression of Otof (green) is significantly decreased in the CKO IHCs, while expression of Sh3gl2, Synj1, Dnm1, and Rab3a (all in green) remains unchanged at the mRNA level. Please note that the mRNA expression levels are quantified by the number, instead of the fluorescence intensity, of the hybridization dots that represent single mRNA molecules. Red: immunofluorescence staining of MYO7A as the hair cell marker. Scale bars: 10 μm. Examined in three mice each in WT and CKO groups. The data are statistically analyzed using unpaired t test.
Fig 4: Characterization of in vitro isolated SVs. (A) SVs enriched by ultracentrifugation, negatively stained by uranyl acetate. White arrows indicate the aggregation of vesicles. Scale bar = 200 nm. (B) SVs collected through home‐made gel filtration column with Sephacryl S‐1000 beads and negatively stained by uranyl acetate. Scale bar = 200 nm. Zoomed bar = 50 nm. (C) SVs isolated with iodixanol layer, negatively stained by uranyl acetate. Scale bar = 200 nm. Zoomed bar = 50 nm. (D) Protein levels of SV2C, vGlut1 and Rab3A were determined via western blotting. (E) SVs labeled with 10‐nm gold tracer, negatively stained by uranyl acetate. Scale bar = 50 nm. (F) The measurement of the diameter of the purified SVs.
Fig 5: 3D mapping of proteins on vesicles with nanogold.a Tomogram z slices of a single clathrin-coated vesicle from peak to the base of the structure. b 3D model view of the vesicle from contour points for membrane (magenta) and scatter points for gold (blue) particles (upper panel, scale bar = 50 nm). Scheme (lower panel) for obtaining positions for gold particles with respect to vesicle membrane contours. Radius for each contour point (magenta, r), its height from the base of the vesicle (Z), radius for gold particle (blue), and its height. A representative profile on the right showing the distribution of gold particles (blue) with respect to the vesicle membrane (red). c Spatially averaged and normalized distribution profile for clathrin, cavin1, EPS15, Rab3a, Rab27a, Rabphilin3a, Granuphilin-a, and Rim2 on vesicles. The particles are reflected across the ordinate. d Density of gold-labeled proteins along the vesicle height (10 bins). The histograms show the sum of particles in each bin (for all vesicles) divided by the average radius of the bin per vesicle. The total number of vesicles collected and analyzed from two independent experiments (for a–d) are: clathrin light chain A, 54; Cavin1, 63; EPS15, 65; Rab3a, 59; Rab27a, 58; Rabphilin3a, 58; Granuphilin-a, 62; Rim2, 63 (Supplementary Table 2).
Supplier Page from Abcam for Anti-Rab3A antibody