Fig. 7
From: Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi
Cargo trafficking out of, but not into, the Golgi is reduced in Vti1a/b-deficient neurons. a Cartoon representing trafficking assay (originally described by ref. 29). Fluorescent cargo fused to SBP (Streptavidin-binding protein) is retained in the ER of KDEL-Streptavidin expressing neurons. Biotin reverses SBP-Streptavidin binding and cargo is free to traffick to the Golgi and, subsequently, outside of this organelle. b, c Representative examples of neurons expressing NPY-SBP-EGFP (b) or GPI-SBP-EGFP (c) and immunostained with MAP2. d Representative time point examples of DHZ (left) and DKO (right) neurons expressing NPY-SBP-EGFP as reporter and ManII-ECFP as Golgi marker. Biotin was added at t = 0. e Fluorescent traces of NPY-SBP-EGFP in the Golgi. f Similar ER-to-Golgi transport of NPY, measured as the fitted slope, between groups (DHZ: 1.83 ± 0.26, n = 12; DKO: 1.68 ± 0.13, n = 14; t-test. g Golgi exit of NPY, measured as the percentage of cargo that leaves the Golgi relative to the peak, at t = 64 (DHZ: 61.36 ± 7.74, n = 12; DKO: 32.61 ± 7.45%, n = 14; Mann–Whitney). h Golgi exit of NPY, measured as the percentage of cargo that leaves the Golgi relative to the peak, at t = 104 (DHZ: 85.49 ± 6.51, n = 12; DKO: 46.27 ± 9.59%, n = 14; Mann–Whitney). i Representative time point examples of DHZ (left) and DKO (right) neurons expressing GPI-SBP-EGFP and MannII-ECFP as Golgi marker. Biotin was added at t = 0. j Fluorescent traces of GPI-SBP-EGFP in the Golgi. k ER-to-Golgi transport of GPI, measured as the fitted slope, is similar between groups (DHZ: 2.66 ± 0.17, n = 15; DKO: 2.57 ± 0.19, n = 18; t-test). l Golgi exit of GPI, measured as the percentage of cargo that leaves the Golgi, 64 min after the ER peak (DHZ: 76.58 ± 5.78, n = 14; DKO: 51.87 ± 4.45%, n = 18; Mann–Whitney). m Golgi exit of GPI, measured as the percentage of cargo that leaves the Golgi, 104 min after the ER peak (DHZ: 94.27 ± 3.66, n = 12; DKO: 84.10 ± 3.83%, n = 18; Mann–Whitney). Bars and trace plots show mean ± SEM. Scatterplots and columns represent individual neurons and independent litters, respectively. **p < 0.01. Scale bar = 2 µm (b, c) and 5 µm (d, h)
