(A) The Golgi apparatus (labelled with galactosyl-transferase fused to mEmerald) rapidly disperses upon BFA (5 μg/ml) application. Scale bar, 15 µm. (B) Distribution of somatic GFP-ERGIC53 before and 15 min after application of BFA. Scale bar, 25 µm. (C) Dendritic segments from neurons expressing GFP-ERGIC53 (top pair) or TfR-mCh (bottom pair) before and 120’ after the application of BFA. Scale bar, 15 µm. Dendrites computationally straightened in ImageJ. (D) Quantification of the number of TfR (red) and ERGIC53 (green) puncta after treatment with BFA (mean ± SEM, n = 7 neurons from 2 independent experiments) plotted on the left axis. The dispersal of fluorescent signal from the somatic Golgi is plotted on the right axis normalized to the pre-BFA value (blue, mean ± SEM, n = 5 neurons from 2 independent experiments). (E) Schematic of the experimental design to investigate Golgi-independent trafficking. Organelle distribution of GluA1 is highlighted in red. Before ER-release, GluA1 is distributed throughout the ER (ER). 2 hr after addition of DDS at 20°C GluA1 accumulates in the dendritic ERGIC and somatic Golgi compartments (ERGIC/Golgi). Addition of BFA disrupts the somatic Golgi, but leaves dendritic ERGIC intact (ERGIC). Cells are returned to 37°C and after a 1 hr incubation, RE-localization is assessed (panels F,G). 2 hr after returning to 37°C, cells are surface labeled to assess membrane delivery (panel H). (F) Shown is a kymograph from a dendritic segment showing cotrafficking between mCh-GluA1 and TfR-GFP in the presence of BFA, 60 min following return to 37 ˚C. (see ‘RE’ panel in diagram from 8E). Black arrow denotes timing of the bleach. Blue arrowheads indicate cotrafficking vesicles. (G) Quantification of cotrafficking between GluA1 vesicles and TfR signal (black bars) and TfR vesicles and GluA1 signal (grey bars) after application of BFA (mean ± SEM, n = 5–6 neurons/condition from 3 experiments, *p=0.041 and n.s. p=0.59 based on unpaired two-tailed Student’s t-test). Numbers on each bar indicate the total number of vesicles. (H) Surface delivery of GluA1 occurs when the GA is disrupted. Surface GluA1 was measured in cells incubated at 20 ˚C, then shifted to 37 ˚C without DDS; cells treated with DDS but maintained at 20 ˚C; cells treated with BFA prior to DDS addition; or cells treated with DDS at 20 ˚C to allow ER release, followed by BFA treatment and 37 ˚C incubation. Values are reported as a percentage of maximal delivery that occurs in neurons treated with DDS, but not treated with BFA; control condition n = 25 neurons. The number of neurons measured for each experimental condition are displayed on the bar graph from 2 independent experiments (values are reported as mean ± SEM; ** p 0.0077, 0.0043 and 0.007 respectively (left to right) by unpaired two-tailed Student’s t-test.). (I) Cortical neuron coexpressing TfR-HaloTag(JF646) and 3xFM-mEOS3.2-GluA1 before (pre) and immediately after dendritic photoconversion (0’) as well as 150 min after addition of DDS. Expanded images (from blue rectangle) showing dendritic mEOS*-GluA1 and TfR-HT localization before and 150 min following ER release are shown to the right. The intensity plots were generated using the dotted orange lines. Blue arrows indicate the overlapping intensity peaks present in both the mEOS*-GluA1 channel and in the TfR channel. Scale bar, 20 μm; Inset scale bar, 5 μm.