Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) Schematic representation of the iFRAP protocol for VSVG or E-cadherin. ( B ) Cells (the images shown were obtained from HeLa cells) were transfected with VSVG-GFP and the cargo was accumulated in the ER at 40 °C overnight. The cells were then shifted to 32 °C for 30 min to allow the cargoes to enter the Golgi. Then the fluorescence in the Golgi area was photobleached, and the cargo was allowed to enter the Golgi area for 5 min followed by photobleaching of the whole cell fluorescence except the Golgi (iFRAP). Bar: 10 μm. ( C ) The exit of the newly arrived VSVG from the Golgi area was then monitored and the fluorescence in the Golgi area was calculated over time and represented as normalized to the initial (after iFRAP) fluorescence levels. In parallel, HeLa cells were transfected overnight with E-cadherin-GFP-RUSH constructs. Biotin was then added to facilitate cargo entry into the Golgi, after which the iFRAP protocol (see ( A )) was used to track the exit of newly arrived E-cadherin from the Golgi. (mean +/− SE; n = >3; see Fig. ). The cell lines and the cargo used for the study are indicated. ( D ) The data from the cargo exit kinetics ( C ) were extracted to analyze only those between 15 min after iFRAP and 40 min (black). Time frame highlighted by the gray square. The data for the VSVG-GFP and E-cadherin-GFP-RUSH exiting from Golgi fit well with a monoexponential decay (red) with the following rate constants: VSVG in HeLa: 0.077 min −1 , A375: 0.138 min -1 , RPE: 0.102 min −1 , COS7: 0.082 min −1 ; E-cadherin in HeLa: 0.01975 min −1 . All five data fit had a R-squared value > 0.99. ( E ) Images from the indicated time points of the cargo exit following iFRAP are shown. Arrowheads indicate post-Golgi carriers. Bar: 10 μm. The adjacent graph compares the exit of cargo from the Golgi (black lines) with the number of carriers present in the cell (pink) over the indicated time period. Carriers represent the fluorescent spots present outside the Golgi area. .

Article Snippet: Polyclonal Rabbit VSVG , Bethyl Laboratories Inc. , Cat #A190-131A; RRID:AB_155862.

Techniques: Transfection, Fluorescence, Construct

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) The individual traces of the iFRAP data presented in Fig. are shown. ( B ) Representative traces from A were normalized in two different ways. As presented in ( A ) or they were normalized by total fluorescence to account for bleaching (orange). The curves were qualitatively similar with flat phase followed by an exponential decrease. ( C ) The intensity levels of background (BG; area with no cells), ER and Golgi were measured before and after bleaching in the iFRAP protocol. The bleaching was efficient in the ER while there was little to no change in the Golgi fluorescence (mean ± SD; n > 3). ( D ) VSVG-GFP-RUSH construct localization with Golgi markers (GM130 and TGN46) from the replicate 2 of Fig. . The positions of VSVG-GFP-RUSH and Golgi marker peaks were normalized, setting GM130 as 0 and TGN46 as 1. Measurements obtained using line scan analysis (red) are compared with those from the GLIM based automated method (blue) within the same experiment. Each data point represents an individual measurement, with results from both methods shown side-by-side for direct comparison. The relative position of the VSVG-GFP-RUSH construct is plotted at each indicated time point (median ± SE; n is indicated in the figure, n.s = not significant; [Student’s t test]). .

Article Snippet: Polyclonal Rabbit VSVG , Bethyl Laboratories Inc. , Cat #A190-131A; RRID:AB_155862.

Techniques: Fluorescence, Construct, Marker, Comparison

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) HeLa cells were transfected with VSVG-GFP-RUSH constructs overnight and then treated with nocodazole (33 μM) for 3 h at 37 °C. Biotin was then added to cells and at indicated times the cells were fixed and stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The relative position of the cargo with respect to GM130 and TGN46 is shown. Bar: 1 μm. ( B ) The position of the cargo and Golgi marker peaks were normalized considering the position of the GM130 peak to be 0 and TGN46 to be 1. The relative positions of cargo peaks over time are plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color). ( C ). HeLa cells were transfected with E-cadherin-GFP-RUSH construct overnight and then treated with nocodazole (33 μM) for 3 h at 37 °C. Biotin was then added to cells and at indicated times the cells were fixed and stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The position of the cargo and Golgi marker peaks were normalized considering the position of the GM130 peak to be 0 and TGN46 to be 1. The relative positions of cargo peaks over time are plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color). ( D ) HeLa cells were co-transfected with VSVG-Halotag and GalT-CFP, and the cargo was accumulated in the ER at 40 °C overnight. Cells were treated with nocodazole (33 μM) for 3 h, then shifted to 32 °C to allow the cargoes to enter the Golgi, and fixed at each indicated time. Cells were then stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The position of the cargo and Golgi marker peaks were normalized considering the position of GM130 peak to be 0 and TGN46 to be 1. The relative positions of cargo peaks over time are plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color. n.s = not significant (*** P = 1.96e-33; 3.2e-07; 6.89161e-07 [Student’s t test]). .

Article Snippet: Polyclonal Rabbit VSVG , Bethyl Laboratories Inc. , Cat #A190-131A; RRID:AB_155862.

Techniques: Transfection, Construct, Staining, Marker

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) HeLa cells were transfected with VSVG-GFP and kept overnight at 40 °C. Then cells were treated with nocodazole for the indicated amount of time and shifted to 32 °C for 25 min and then fixed and prepared for immunofluorescence with the indicated markers. White boxes represent insets. Bar: 10 μm. ( B ) Quantification from ( A ) of the number of ministacks showing the indicated markers. Mean +/− SD. Data points ( n > 290) from 3 independent experiments. ( C ) The Pie chart shows the percentage of VSVG-mCherry-containing tubules exiting from TGN and those whose origin is unclear ( n = 13). We have not observed any tubule originating from cis -Golgi. ( D HeLa cells were transfected with VSVG-mCherry-RUSH construct overnight and then biotin was added for 1 h. The cells were fixed and stained for GM130 and TGN46 to mark the cis -Golgi and TGN respectively. White boxes represent insets. Bar: 5 μm. .

Article Snippet: Polyclonal Rabbit VSVG , Bethyl Laboratories Inc. , Cat #A190-131A; RRID:AB_155862.

Techniques: Transfection, Immunofluorescence, Construct, Staining

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: Upper panel: Cells treated as in Fig. (except for the nocodazole treatment) were fixed and prepared for EM. The presence of VSVG-GFP was visualized by anti-GFP antibody pointed with red arrowsheads (10 nm gold particles) and GM130 to indicate cis -Golgi with anti-GM130 antibodies circled in blue (5 nm gold particles). Lower panel: The distribution of VSVG-GFP gold particles across the Golgi stack was quantitated and presented as a percentage of gold particles per cisterna (see “Methods”) (mean +/− SE; Data points n > 15 stacks). .

Article Snippet: Polyclonal Rabbit VSVG , Bethyl Laboratories Inc. , Cat #A190-131A; RRID:AB_155862.

Techniques:

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) HeLa Cells were transfected with VSVG-GFP and kept overnight at 40 °C. Then cells were shifted to 32 °C for 1 h and then fixed and prepared for EM. The sections were labeled for GFP (15 nm gold) and TGN46 (10 nm gold; indicated by blue circles). Bar: 100 nm. ( B ) HeLa Cells were transfected with VSVG-GFP-RUSH, GPI-GFP-RUSH or LAMP1-GFP-RUSH constructs and kept at 37 °C. Then cells were incubated with 1 μM biotin for 60 min at 37 °C. The cells were fixed and stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The relative position of the cargo relative to the GM130 and TGN46 is shown. Bar: 2 μm. ( C ). In HeLa cells treated as in ( B ), the position of each indicated RUSH construct and Golgi marker peaks were normalized, considering the position of the GM130 peak to be 0 and TGN46 to be 1. The relative position of each indicated RUSH construct peak at 60 min is plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color). ( D ) HeLa cells were transfected with E-cadherin-GFP-RUSH constructs overnight and then treated with nocodazole (33 μM) for 3 h at 37 °C. The cells were treated with cycloheximide for the last 1 h at 37 °C. Biotin was then added to cells to allow the cargoes to enter the Golgi ministacks for 60 min. Then the fluorescence in the cell, except for a few Golgi ministacks, was photobleached (iFRAP). Left panel: the exit of the cargoes from the Golgi ministack was then monitored, and the fluorescence in the Golgi area was calculated over time and represented as normalized to the initial (after iFRAP) fluorescence levels (mean +/− SE; n = 2; 5 ministacks). Right panel: the data for the E-cadherin-GFP-RUSH exiting from Golgi ministacks fit well with a monoexponential decay (red) with the following rate constants: 0.005839 min -1 , with an R-squared value > 0.97. ( E ) Schematic representation of ( C ) of the iFRAP protocol for E-cadherin in cells treated with nocodazole. .

Article Snippet: Polyclonal Rabbit VSVG , Bethyl Laboratories Inc. , Cat #A190-131A; RRID:AB_155862.

Techniques: Transfection, Labeling, Construct, Incubation, Staining, Marker, Fluorescence