Figure 4. Dendritic Golgi satellites localize near endoplasmic reticulum exit sites (ERESs) and endosomes.

(A) Golgi satellites (St3-GFP) frequently pair with ERESs (mChSec 23) in dendrites. Primary cortical cultures were transfected with St3-GFP, mCh-Sec23, and HA-tagged α4β2R subunits. Neurons were treated with nicotine for 17 hr and fixed. Dendrites were imaged for Golgi satellites (St3-GFP, green) and ERESs (mChSec 23, red). Closely aligned Golgi satellites and ERESs are marked by yellow arrows. Scale bar, 5 µm. (B) An example of the close association between Golgi satellites (green) and ERESs (red). Signal intensity of the Golgi satellite and ERESs measured in arbitrary fluorescent units (AFU) in a line scan through the pair in the boxed area. (C) Golgi satellites (St3-GFP) frequently pair with early endosomes (EEA1 staining) in dendrites. Neurons were transfected with St3-GFP and after nicotine treatment, fixed and stained with antibodies to EEA1 before imaging as in A. Golgi satellites closely aligned with early endosomes are marked by yellow arrows. Scale bar, 5 µm. (D) An example of the close association between Golgi satellites (green) and early endosomes (red). Signal intensity of the two compartments was measured and displayed as in A. (E) Golgi satellites (St3-Halo) frequently form triads with ERESs (mChSec 23) and early endosomes (EEA1 staining) in dendrites. Neurons were transfected with St3-GFP, mCh-Sec23, and treated with nicotine for 17 hr before fixation and antibody labeling for EEA1 and imaging. Golgi puncta (green) closely aligned with ERESs (red) and early endosomes (mauve) are marked by yellow arrow heads. Scale bar, 10 µm. (F) An example of the close association between Golgi satellites (green), ERESs (red), and early endosomes (mauve). Signal intensity for markers of the three organelles was measured and displayed as in A and C.

Figure 4—figure supplement 1. Moderate exogenous expression of the Golgi enzymes, Man II-GFP, GalNac-T2-mCherry, and St3-Halo in pairs, or singly, along with labeling for internalized fluorescently tagged wheat germ agglutinin (WGA) or an St3 antibody.

(A) Cortical cultures were transfected with the indicated pairs of probes or were singly transfected and then either surface-labeled with WGA-Alexa 488 for 20 min, washed, then incubated at 37°C for 2 hr, or stained with a polyclonal antibody (pAb) against St3. Scale bar, 5 µm. (B) Quantification of the percentage of Golgi enzyme puncta that co-localized with different Golgi markers. Data are shown as mean ± SEM, St3-Halo and St3 pAb, 76.3 ± 4.9; St3-Halo and WGA, 85.8 ± 4.5; St3-Halo and Man II-GFP, 74.7 ± 3.6; GalNac-T2-mCherry and Man II-GFP, 77.4 + 3.9; GalNac-T2-mCherry and St3 pAb, 48.3 + 4.2. (n = 8–11 fields per group).

Figure 4—figure supplement 2. Measuring the punctal overlap between endogenous or expressed St3, and the endosomal markers, EEA1, internalized transferrin receptor, or VPS35.

(A) Cortical cultures fixed and stained with the St3 polyclonal antibody (pAb) or transfected with St3-Halo for 48 hr, were co-labeled with antibodies against EEA1 (early endosomes), VPS35 (retromer), or surface-internalized Tfr-Alexa 568 (recycling endosomes; cultures were live-labeled for 30 min, washed, and cells incubated at 37°C for 1 hr). Scale bar, 5 µm. (B) Quantification of the percentage of Golgi enzyme puncta that co-localized with different endosomal markers. Data are shown as mean ± SEM, St3-Halo and EEA1, 26.4 ± 4.8; St3-Halo and TfR, 19.4 ± 7.4; St3 pAb and EEA1, 22.6 ± 4.0; St3 pAb and TfR, 28.9 + 3.8; St3 pAb and VPS35, 19.9 + 2.6 (n = 5–10 fields per group).