Figure 3.
Figure 3. The stabilization of ERTGoCS decreases PI4P at the TGN in a FAPP1-dependent manner. (A) Schematic representation of the opto-ERTGoCS construct. (B) The optogenetic approach used to visualize ERTGoCS. In the absence of light (left), the ER-fused SsrA domain is enclosed in the C-terminal helix of the LOV domain (iLID construct). Blue light activation (488 nm, right) induces a conformational change that releases the SsrA domain, allowing its dimerization with the TGN-fused SspB domain and, in turn, ERTGoCS stabilization. (C) Representative images of HeLa cells transfected with the opto-ERTGoCS construct and with the PI4P probe P4M (PI4P). Cells were kept in the dark (left panels) and pulsed with blue light (488 nm; middle panels), and then the blue light was removed (right panels). After blue light stimulation, Cb5 is recruited to the TGN46-GFP area (inset, middle panels) and P4M Golgi localization is reduced. After removal of blue light, Cb5 returns to the ER localization, and P4M intensity increases in the Golgi area. Bar, 10 µm. (D) Mean fluorescence intensity values ± SD of Cb5-mCherry (red) and P4M (gray) in the Golgi area (defined as TGN46-GFP localization; see Materials and methods) in control (left) and in FAPP1-KD cells (right). *, P < 0.05. (E) mCherry-LOV-SsrA-Cb5 localization and PI4P Golgi levels (detected by anti-PI4P antibody) in HeLa cells transfected with the opto-ERTGoCS construct in the dark (top panels), exposed to a blue-light pulse (middle panels) and after removal of light stimulus (bottom panels). Bar, 10 µm. (F) Quantification of the ratio of PI4P and Golgin-97 fluorescence intensity in the Golgi area expressed as a percentage of maximum. Three independent experiments, n = 50. ***, P < 0.001; Student’s t test. (G and H) Schematic representation of constructs used to stabilize the ERTGoCS with rapamycin. (I) PI4P distribution in control, VAP-KD, FAPP1-KD, and VAP-KD+FAPP1-KD cells expressing TGN46-FRB-HA and mCherry-T2A-FKBP-Cb5 reporter proteins upon short (2 min rapamycin, middle panel) and prolonged (15 min rapamycin, right panel) stabilization of ERTGoCS. Inserts show Cb5 localization. Arrowheads indicate ERTGoCS formation. Bar, 10 µm. (J) Quantification of Golgi PI4P levels (solid lines) and number of cells forming ERTGoCS (dashed lines) in control, VAP-KD, FAPP1-KD, and VAP-KD+FAPP1-KD cells treated with 200 nM rapamycin for the indicated times. PI4P was detected using a monoclonal anti-PI4P antibody, and PI4P levels were calculated as the ratio between PI4P and Golgin-97 fluorescence intensity at the Golgi complex. PI4P values are expressed as a percentage of values in cells at time 0 (not exposed to rapamycin). ERTGoCS values are expressed as a percentage of cells showing colocalization of Cb5 with TGN46. Means ± SD, three independent experiments; n = 60–100 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test. (K) Golgi PI4P levels (solid lines) and number of cells forming ERTGoCS (dashed lines) in HeLa WT and VAP-KO cells treated with 200 nM rapamycin for the indicated times. PI4P levels were calculated as the ratio between PI4P and Golgin-97 fluorescence intensity at the Golgi complex. PI4P values are expressed as a percentage of values at time 0 (cells not exposed to rapamycin). ERTGoCS values are expressed as a percentage of cells showing colocalization of Cb5 with TGN46. Means ± SD, three independent experiments; n = 60–100 cells; Student’s t test.

The stabilization of ERTGoCS decreases PI4P at the TGN in a FAPP1-dependent manner. (A) Schematic representation of the opto-ERTGoCS construct. (B) The optogenetic approach used to visualize ERTGoCS. In the absence of light (left), the ER-fused SsrA domain is enclosed in the C-terminal helix of the LOV domain (iLID construct). Blue light activation (488 nm, right) induces a conformational change that releases the SsrA domain, allowing its dimerization with the TGN-fused SspB domain and, in turn, ERTGoCS stabilization. (C) Representative images of HeLa cells transfected with the opto-ERTGoCS construct and with the PI4P probe P4M (PI4P). Cells were kept in the dark (left panels) and pulsed with blue light (488 nm; middle panels), and then the blue light was removed (right panels). After blue light stimulation, Cb5 is recruited to the TGN46-GFP area (inset, middle panels) and P4M Golgi localization is reduced. After removal of blue light, Cb5 returns to the ER localization, and P4M intensity increases in the Golgi area. Bar, 10 µm. (D) Mean fluorescence intensity values ± SD of Cb5-mCherry (red) and P4M (gray) in the Golgi area (defined as TGN46-GFP localization; see Materials and methods) in control (left) and in FAPP1-KD cells (right). *, P < 0.05. (E) mCherry-LOV-SsrA-Cb5 localization and PI4P Golgi levels (detected by anti-PI4P antibody) in HeLa cells transfected with the opto-ERTGoCS construct in the dark (top panels), exposed to a blue-light pulse (middle panels) and after removal of light stimulus (bottom panels). Bar, 10 µm. (F) Quantification of the ratio of PI4P and Golgin-97 fluorescence intensity in the Golgi area expressed as a percentage of maximum. Three independent experiments, n = 50. ***, P < 0.001; Student’s t test. (G and H) Schematic representation of constructs used to stabilize the ERTGoCS with rapamycin. (I) PI4P distribution in control, VAP-KD, FAPP1-KD, and VAP-KD+FAPP1-KD cells expressing TGN46-FRB-HA and mCherry-T2A-FKBP-Cb5 reporter proteins upon short (2 min rapamycin, middle panel) and prolonged (15 min rapamycin, right panel) stabilization of ERTGoCS. Inserts show Cb5 localization. Arrowheads indicate ERTGoCS formation. Bar, 10 µm. (J) Quantification of Golgi PI4P levels (solid lines) and number of cells forming ERTGoCS (dashed lines) in control, VAP-KD, FAPP1-KD, and VAP-KD+FAPP1-KD cells treated with 200 nM rapamycin for the indicated times. PI4P was detected using a monoclonal anti-PI4P antibody, and PI4P levels were calculated as the ratio between PI4P and Golgin-97 fluorescence intensity at the Golgi complex. PI4P values are expressed as a percentage of values in cells at time 0 (not exposed to rapamycin). ERTGoCS values are expressed as a percentage of cells showing colocalization of Cb5 with TGN46. Means ± SD, three independent experiments; n = 60–100 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test. (K) Golgi PI4P levels (solid lines) and number of cells forming ERTGoCS (dashed lines) in HeLa WT and VAP-KO cells treated with 200 nM rapamycin for the indicated times. PI4P levels were calculated as the ratio between PI4P and Golgin-97 fluorescence intensity at the Golgi complex. PI4P values are expressed as a percentage of values at time 0 (cells not exposed to rapamycin). ERTGoCS values are expressed as a percentage of cells showing colocalization of Cb5 with TGN46. Means ± SD, three independent experiments; n = 60–100 cells; Student’s t test.

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