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. 2024 Mar 11;12:RP91708. doi: 10.7554/eLife.91708

Figure 1. TGN46 is required for cargo sorting and loading into CARTS.

(A) Pancreatic adenocarcinoma upregulated factor (PAUF) secretion assay in parental (WT) and CRISPR/Cas9-mediated TGN46 knockout (KO) HeLa cells. Cells expressing PAUF-MycHis were incubated for 4 hr in fresh medium without serum in the absence (Ctrl) or presence of Brefeldin A (BFA), after which the cells were lysed. Equal amounts of cell lysates and their corresponding secreted fraction (medium) were analyzed by Western blotting (left) using an anti-Myc antibody (top blot) and anti-TGN46 antibody (bottom blot). BFA was included as a positive control of secretion inhibition. Quantification (right) of the ratio of secreted/internal PAUF signal normalized (norm.) as a percentage to the average value of the WT, Ctrl condition. n = 4 independent experiments. Individual values shown with connecting lines between paired datasets (shown only for untreated condition, for clarity), with mean ± stdev. Paired t test (*p ≤ 0.05; **p ≤ 0.01). (B) Number of carriers (CARTS, empty circles; VSVG carriers, gray circles) per unit area observed in WT or TGN46-KO HeLa cells expressing either PAUF-mRFP (CARTS marker) and VSVG-HA (VSVG carrier marker). At least 10 cells from each of n = 3 independent experiments were quantified. Individual values shown, with mean ± stdev. Unpaired two-tailed t test (ns, p > 0.05; *p ≤ 0.05). (C) Relative fluorescence intensity average time trace (mean ± standard error of the mean [s.e.m.]) of fluorescence loss in photobleaching (FLIP) experiments performed in WT or TGN46-KO HeLa cells expressing PAUF-mRFP. (D) Quantification of the PAUF-mRFP Golgi residence time as obtained from the FLIP experiments, as the one shown in (C). Between 7 and 12 cells from each of n = 3 independent experiments were quantified. Individual values shown, with mean ± stdev. Unpaired two-tailed t test (***p ≤ 0.001). (E) Left panels are confocal fluorescence microscopy images of HeLa cells transfected with control (Ctrl) or TGN46 siRNA, which were also transfected with PKD2-KD-Flag and PAUF-mRFP. Cells were fixed at steady state and labeled with anti-TGN46 (cyan) and anti-Flag (magenta) antibodies. PAUF-mRFP fluorescence signal is shown in green. Scale bars are 10 µm, magnifications of the boxed regions are shown. In the right panel, the quantification of the percentage of PKD2-KD-induced tubules that containing PAUF-mRFP in control (Ctrl) or TGN46 siRNA-treated cells. At least 10 cells from each of n = 3 independent experiments. Individual values shown, with mean ± stdev. Unpaired two-tailed t test (****p ≤ 0.0001).

Figure 1—source data 1. Uncropped images of the membranes of the Western blotting shown in Figure 1A.

Figure 1.

Figure 1—figure supplement 1. TGN46 is required for cargo sorting and loading into CARTS.

Figure 1—figure supplement 1.

(A) HeLa WT or HeLa TGN46-KO cells expressing PAUF-mRFP were fixed, processed for immunostaining, and the localization of endogenous TGN46 and PAUF-mRFP was monitored by immunofluorescence microscopy. (B) HeLa WT or HeLa TGN46-KO cells expressing VSVG-HA and PAUF-mRFP were fixed, processed for immunostaining, and the localization of VSVG-HA and PAUF-mRFP was monitored by immunofluorescence microscopy. (C) Percentage of PAUF-mRFP-containing carriers (CARTS) that are also positive for VSVG-HA in HeLa WT or TGN46-KO cells, quantified from confocal micrographs as those shown in (B). Results are from at least 10 cells from each of n = 3 independent experiments (individual values shown, with mean ± stdev). Unpaired two-tailed t test (*p ≤ 0.05). (D) Top: Schematic representation of fluorescence loss in photobleaching (FLIP) experiments (see text and methods for details). Bottom: Fluorescence microscopy images obtained from a characteristic FLIP experiment assessing the export rate of PAUF-mRFP from the perinuclear area in either HeLa WT or HeLa TGN46-KO cells. Time from the beginning of the FLIP experiments is indicated, and the area enclosed by the white lines shown in the left images denotes the photobleached area. (E) Quantification of the number of PKD2-KD-containing tubules per cell in HeLa cells transfected with control (Ctrl) or TGN46-targeting siRNA. Results are from at least 10 cells from each of n = 3 independent experiments (individual values shown, with mean ± stdev). Unpaired two-tailed t test (ns, p > 0.05). Scale bars in (A, B, D) are 10 µm.