Supplemental Materials

This article contains the following supporting material:

• Supplementary Figure 1. - (A) Propanolol, U73122, D609 and FB1 alter neither microtubule nor actin cytoskeleton organization. Vero cells were treated with propanolol (60 µM/30 min), U73122 (6 μM/30 min), D609 (500 µM/30 min), or FB1 (25 g·ml^-1/24 h) fixed and processed for immunofluorescence microscopy using antibodies to ß-tubulin to visualize microtubules, to filamentous actin organization (F-actin) with phalloidin-TRITC, or to giantin to view the Golgi complex. (B) Unlike D609, neither propanolol nor U73122 nor FB1 alter microtubule dynamics. Vero cells were treated first with the microtubule-disassembly agent nocodazole (NZ; 30 µM/2 h) and subsequently with propanolol, U73122, D609 or FB1 in the continuous presence of NZ for a further 15 min. This treatment completely disassembled microtubules. Thereafter, NZ was washed out and the microtubule reassembly was examined for different times until 2 h, in the continuous presence of propanolol, U73122, D609 or FB1. Cells were fixed and processed for immunofluorescence microscopy using antibodies to ß-tubulin. Unlike propanolol U73122 and FB1, D609 prevented the normal reassembly of microtubules after NZ wash out. (C) Propanolol and U73122 and FB1 do not affect actin dynamics. In another set of experiments, cells were treated with LtB (500 nM/30 min). Subsequently, cells were incubated with propanolol or U73122 for 15 min in the continuous presence of LtB. Thereafter, LTB was washed out and cells fixed and stained with rodamine-phalloidin at different times to visualize the kinetics of the F-actin reassembly. Neither the disassembly not the reassembly of F-actin organization was affected by propanolol, U73122 or FB1 treatments. Bars, 10 μm.
• Supplementary Figure 2. - Propanolol, U73122 and FB1 do not alter the ER-to-Golgi VSV-G transport but differently affect its trafficking to the plasma membrane. Vero cells infected with the VSV ts045 temperature-sensitive mutant virus were incubated at non-permissive temperature (40 ºC) where the G glycoprotein was retained in the ER (A, D, G, J). At permissive temperature (32 ºC), both in untreated and in treated cells, the VSV-G was seen in the Golgi after 15 min (B, E, H, K). After 60 min, VSV-G was retained in the Golgi in cells treated with propanolol (F) or FB1 (L). In contrast, in cells treated with U73122, a significant amount of VSV-G already reached the plasma membrane (I). Bar, 10 μm.
• Supplementary Figure 3. - (A) Fumonisin B1 does not alter the ER/Golgi interface membrane flow. Disassembly and reassembly of the Golgi complex in the presence or after the removal of BFA in untreated- and FB1 (25 μg·ml^-1/24 h)-treated Vero cells. In the presence of BFA, the ER-like staining pattern of giantin appeared independently of the presence of FB1. After BFA wash out, the Golgi complex reassembled normally despite the continuous presence of FB1. The Golgi complex morphology revealed by the giantin staining was unaffected by FB1 treatment. (B) Fumonisin B1 redistributes the C1 domain to the cytoplasm. Control and FB1 (25 μg/ml for 24 h)-treated GFP-C1b domain-transfected Vero cells were fixed and co-stained with anti-Golgi CTR433 protein antibodies. Untreated cells showed a preponderant Golgi localisation of the GFP-C1 domain whereas in FB1-treated cells the GFP fluorescence was completely redistributed to the cytoplasm. Bar, 10 μm.
• Supplementary Figure 4. - Additive effects of propanolol and U73122 on the subcellular distribution of the KDELr. Vero cells were treated with propanolol (40 µM/60 min; A) or U73122 (4 µM/60 min; B) they showed a normal staining pattern of KDELr (compare these two panels with that shown in Fig. 3E). However, when propanolol and U73122 (at same concentrations) were added together, the staining pattern for the KDELr was more Golgi-like as a consequence of the decrease in the density of fluorescent punctate cytoplasmic structures (C), whose immunofluorescence staining pattern was indistinguishable from that seen in propanolol (60 μM) or U73122 (6 μM)-treated cells alone (compare with Fig. 3F and G, respectively). Bar, 10 μm.
• Supplementary Figure 5. - Propanolol and U73122 interfere neither with the steady state distribution of β-COP nor with its release from the Golgi induced by BFA. Control (A and B), propanolol (60 μM/5 min; C and D)-, or U73122 (6 μM/5 min)-treated Vero cells were fixed and co-stained for the Golgi matrix protein GM130 and β-COP. Note that propanolol and U73122 did not release β-COP by themselves (D and F, respectively). Control, propanolol- and U73122-treated cells were subsequently incubated with BFA (5 μg/ml for 3 min), fixed and processed for immunofluorescence microscopy. Both in control, propanolol and U73122-treated cells, β-COP was normally redistributed from the Golgi to the cytosol in the presence of BFA (G, H, K, respectively). Bar, 10 μm.
• Supplementary Figure 6. - DOG prevents the inhibitory effect of propanolol and U73122 on the BFA-induced disassembly of the Golgi complex. Vero cells were treated with BFA (5 µg/ml for 20 min) alone, with propanolol (60 μM) plus BFA (propanolol was added 5 min before BFA) or U73122 (6 μM) plus BFA (U73122 was added 5 min before BFA) as indicated and shown in Fig. 3 (A-C). In parallel experiments to that shown in Fig. 3 (A-C), cells were incubated with DOG (3 μM) 15 min before the treatment with BFA. Cells were then processed for immunofluorescense microscopy using monoclonal anti-CTR433 to visualize the Golgi complex. DOG treatment did not affect the characteristic disassembly of the Golgi complex induced by BFA (compare B with Fig. 3A). In contrast, DOG pre-treatment significantly prevented the inhibition caused by propanolol (compare C with Fig. 3B)- or U73122 (compare D with Fig. 3C) on the BFA-induced Golgi disassembly. (E) Quantitative analysis of results. Statistical significance, p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***).Bar, 10 μm.
• Supplementary Figure 7. - The Golgi localisation of CtBP3/BARS is not altered by propanolol or propanolol plus U73122. Control (A), propanolol (60 μM/30 min) (B) or propanolol (60 μM/30 min) plus U73122 (6 μM/30 min) (C) SLO-permeabilised Vero cells were stained with a monoclonal antibody to BARS. Bar, 10 μm.
• Video 1. - BFA-induced Golgi tubulation in HeLa cells constitutively expressing YFP-Galactosyltransferase (GalT).
• Video 2. - The BFA-induced Golgi tubulation is not completed in propanolol-treated HeLa cells constitutively expressing YFP-GalT.
• Video 3. - GFP-C1b PKCθ-transfected COS-1 cells in response to PMA (250 nM). At steady-state the GFP-C1 domain is localised to the Golgi. When PMA is added, the GFP-C1 domain is quickly translocated from the Golgi to the plasma membrane.
• Video 4 - GFP-C1b PKCθ-transfected COS-1 cells in response to PMA (250 nM) plus DOG (3 μM). Cells were treated first with PMA and DOG was added after 5 min. In contrast to video 3 (PMA alone), the GFP-C1 domain returns to the Golgi soon after DOG is added.
• Video 5. - GFP-C1b PKCθ-transfected COS-1 cells in response to propanolol (60 μM). When propanolol is added, the Golgi-associated GFP-C1b domain partially translocates to the cytosol, for a short time and then returns to the Golgi.
• Video 6. - GFP-C1b PKCθ-transfected COS-1 cells in response to DOG (3 μM) plus propanolol (60 μM). DOG pre-treatment prevents the propanolol-induced cytosolic translocation of the GFP-C1b (video 5), which remains in the Golgi in spite of the presence of propanolol.
• Video 7. - Dual-axis electron tomogram from a 250 nm semithin section containing a representative Golgi stack of an NRK cell treated with propanolol (60 μM/15 min).
• Video 8. - 3D model of a representative Golgi stack of a propanolol-treated NRK cell shown in video 7. Note numerous COP-coated transport carriers that remain attached to cisterna.
• Video 9. - GFP-ARFGAP1-transfected COS-1 cells treated with propanolol (60 ìM). Golgi-associated fluorescence from GFP-ARFGAP1 is lost after propanolol addition (see Fig. 10 for quantitative analysis).
• Video 10. - GFP-ARFGAP1-transfected COS-1 cells pre-treated with DOG (3 μM) and then with propanolol (60 μM). The propanolol-induced reduction of the Golgi-associated fluorescence from GFP-ARFGAP1 is partially prevented by DOG pre-treatment (see Fig. 10 for quantitative analysis).