(A, B) HeLa cells were fixed 72 h after transfection of control scrambled (A) and cPLA₂α-specific (B) siRNAs and stained for cPLA₂α and giantin. Confocal microscopy shows a strong reduction in cPLA₂α labelling in cells treated with the specific siRNAs (B). (C) HeLa cells treated with siRNAs as in (A) and (B) and prepared for western blotting with antibodies against cPLA₂α and actin. Expression of cPLA₂α was strongly inhibited, while actin levels remained unaffected. (D) Quantification of cPLA₂ activity (mean±SD; measured using [³H]-AA release; see Materials and Methods) revealed its reduction in cPLA₂α-siRNAs-treated HeLa cells. (E, F) Control and cPLA₂-specific siRNAs-transfected HeLa cells. Labelling with an anti-giantin ab (E) and morphometric analysis (F) show extensive fragmentation of the Golgi complex in cells with low cPLA₂α expression (E, asterisks). (G–I) Control (G, I) and cPLA₂α-siRNAs-treated HeLa cells (H, I) were fixed and prepared for EM analysis. Tubular structures were seen to connect the Golgi stacks to each other (G, arrows) in control cells but were lost upon cPLA₂α knock-down (H). Morphometric analysis indicates a reduction in surface area (mean±SD; n = 30 stacks) associated with tubular structures in the Golgi complex in cPLA₂α-siRNAs-treated cells (I). (J) HeLa cells transfected with cPLA₂α-GFP, fixed and processed for immono-gold EM with an anti-GFP ab. Cells overexpressing cPLA₂α-GFP show numerous long tubular profiles in the Golgi region (arrows) and partial loss of stack organization. Scale bar, 45 µm (A, B), 22 µm (E), 300 nm (G, H, J).

(A–E) Control (A, B, D) and cPLA₂α-siRNAs-treated HeLa cells (A, C, E) were infected with VSV and kept at 40°C for 3 h to accumulate VSVG within the ER. The cells were fixed directly at the end of the block (A) or after a 60-min (A) or 45-min (B–E) temperature shift to 32°C. (A) The cells were labelled with an anti-VSVG ab, showing that after accumulation within the ER, VSVG was efficiently exported to the plasma membrane in control cells and blocked in the Golgi complex in cPLA₂α-siRNAs-treated cells. (B–E) The cells were triple labelled with anti-VSVG, anti-cPLA₂α, and anti-TGN46 antibodies (B, C) or prepared for immuno-EM using the nanogold protocol (D, E). In control cells, VSVG showed good colocalization with TGN46 in the Golgi area (B, inset); this colocalization was poor in cPLA₂α-siRNAs-treated cells (C, inset). In control cells, EM showed little VSVG in the cis portion of the stack (7.8%), with most (61%) within trans-Golgi compartments (D, arrows), post-Golgi carriers (D, filled arrowhead), and at the plasma membrane (D, empty arrowhead). In cPLA₂α-siRNAs-treated cells, most of the VSVG (62%) remained within the swollen cis portion of the stack (E, arrows). (F, G) Control and cPLA₂α-siRNAs-treated HeLa cells infected with VSV were metabolically labelled with [³⁵S]-methionine and chased at 32°C. At the indicated times, the cells were solubilized and digested with endoglycosidase H (Endo-H), which cleaves sugar chains built on the proteins early in the secretory pathway (i.e., before their processing by the medial Golgi enzyme mannosidase-II, which convert sugars into an Endo-H resistant form). The cell lysates were then separated by SDS-PAGE, and the gels scanned (F). The percentages of the Endo-H-resistant form of VSVG with respect to the total amounts of VSVG were quantified (G) using a FUJIFILM imager. The data indicate that VSVG processing to its Endo-H resistant form (which occurs in the medial Golgi) was reduced when cPLA₂α was silenced. (H) cPLA₂α-siRNAs-treated cells were infected with VSV, microinjected with recombinant cPLA₂α during the 40°C block, and fixed 45 min after the block release at 32°C. The cells were then stained with anti-VSVG and anti-cPLA₂α antibodies and observed under the confocal microscope. VSVG was delivered to the plasma membrane after cPLA₂α microinjection (arrows) but remained in the Golgi in noninjected cells (asterisks). (I) HeLa cells were transfected with VSVG-GFP and the dominant-negative cPLA₂α(1–522) isoform, subjected to a 40°C block, and fixed 45 min after the temperature shift to 32°C. Confocal images reveal VSVG-GFP blocked in the Golgi complex in cPLA₂α(1–522)-expressing cells (asterisks). Scale bar, 60 µm (A), 6 µm (B, C), 270 nm (D), 200 nm (E), 15 µm (H), 8.5 µm (I).

(A, B) Control (A) and cPLA₂α-silenced (B) HeLa cells were treated with 30 µM NZ for 3 h, then fixed and prepared for EM tomography. The digital slices extracted from the tomograms (see Videos S4 and S5) show an intercisternal connection (arrow) bridging cisternae located at the different levels of the stack in control cells (A) and reveals no bridges between cisternae within the Golgi ministack in silenced cells (B). (C, D) Control (C) and cPLA₂α-siRNAs-treated HeLa cells (D) were infected with VSV and kept at 40°C for 3 h in the presence of NZ to disassemble Golgi into ministacks and accumulate VSVG within the ER. The cells were fixed directly at the end of the block or at indicated times after the temperature shift to 32°C. The cells were double labelled with an anti-VSVG and either GM130 (cis-Golgi marker) or TGN46 (trans-Golgi marker) and investigated under confocal microscopy. In control cells (C), after release of 40°C block, VSVG moved from the ER to the Golgi ministacks where it first colocalized with GM130 and then with TGN46 (see insets) and finally arrived to the cell surface (arrows). In contrast, cPLA₂α-silenced cells show VSVG within the GM130-positive compartment of ministacks (see insets) even 60 min after release from the ER, with little or no VSVG detected both in the TGN46-positive compartment and at the plasma membrane. Scale bar, 150 nm (A, B), 6 µm (C, D).

(A–P) IMLFs from cPLA₂α KO mice (IMLFs^−/−) were incubated with siRNAs specific for the different cytosolic PLA₂ enzymes, and control siRNAs, for 72 h (A–P; as indicated). The cells were then infected with VSV, kept at 40°C for 3 h to accumulate VSVG in the ER, and incubated in fresh medium at 32°C for 60 min before fixing. The fixed cells were either stained with only an anti-VSVG ab (A–O) or double labelled for VSVG and GM130 (P). VSVG was efficiently exported to the plasma membrane in cells incubated with either control (O) or most of the PLA₂-isoform-specific siRNAs (A–K, M–O). However, RNAi of GVIIIA-PLA₂ induced accumulation of VSVG within the Golgi complex of IMLFs (L). Notably, VSVG always overlapped strongly with GM130 (see inset in P) in cells incubated with GVIIIA-PLA₂-specific siRNAs, suggesting that VSVG is trapped within the cis-Golgi compartment. (R) IMLF^−/− and control lung fibroblasts from mice expressing cPLA₂α (IMLFs^+/+) were incubated with siRNAs specific for either cPLA₂α or GVIIIA-PLA₂ and control siRNAs for 72 h. The cells were then infected with VSV, subjected to the 40°C block with its further release for 60 min and fixed. VSVG was detected at the surface of IMLFs^−/− and IMLFs^+/+ using an ab against its ectodomain. Afterwards, the cells were permeabilized and incubated again with an anti-VSVG ab to reveal the total pool of VSVG within the cell. Then fluorescense intensities of surface and total VSVG were evaluated, expressed as a ratio, and normalized to the control. This quantification reveals that cPLA₂α silencing significantly inhibits VSVG transport in IMLFs^+/+ (but not in IMLFs^−/−), while GVIIIA-PLA2 RNAi strongly affects VSVG transport in IMLFs^−/− and only slightly in IMLFs^+/+. Scale bar, 18 µm (A–O), 7.2 µm (P).

Inset 1: cPLA₂α hydrolyses the fatty acids (FA) at the sn-2 position of cylindrical phospholipids to form wedge-shaped lysophospholipids. Inset 2: This formation of wedge-shaped lysophospholipids favours generation of spontaneous membrane curvature and transformation of flat cisternae-like membranes into highly curved tubular membranes, which serve as intermediates for transport across the Golgi stack.

(A, B) HFs were incubated for 3 h at 40°C (A) and then shifted to 32°C for 30 min in the presence of ascorbic acid (B), or HFs (C) and HepG2 cells (D) were grown under steady-state conditions. After fixing, the cells were double labelled with antibodies against PC-I and cPLA₂α and examined under the confocal microscope. (A) When PC-I was trapped in the ER after the 40°C block, cPLA₂α enrichment was not detected in the Golgi area. (B) Arrival of PC-I at the Golgi complex after release of the temperature block induced cPLA₂α binding to the Golgi complex. (C, D) Under steady-state conditions, the cells were fixed, labelled with antibodies against cPLA₂α and either PC-I (C) or giantin (D). Confocal microscopy reveals that here some cPLA₂α can be seen in the Golgi area together with PC-I (C) and giantin (D). (E) Fluorescence intensity of cPLA₂α in the Golgi region (defined as giantin-positive area) was quantified and normalized to cPLA₂α intensity in the cytosol of HFs. Plot shows that Golgi/cytosol ratio of cPLA₂α (mean±SD; n = 20 cells) decreases in cells subjected to 40°C transport block, but increases over the levels detected at steady-state conditions upon block release (at 32°C). (F, G) HeLa cells were infected with VSV and kept at 40°C for 3 h to accumulate VSVG in the ER. The cells were then fixed (F) or shifted to 32°C for 30 min to allow VSVG to exit from the ER (G), and prepared for confocal microscopy. Immunofluorescence labelling shows a diffuse pattern of cPLA₂α when VSVG is blocked in the ER (F). In contrast, cPLA₂α undergoes recruitment to the Golgi membranes as soon as VSVG arrives at the Golgi complex from the ER (G). (H) HeLa cells were transfected with cDNA encoding full length cPLA₂α fused with GFP (cPLA₂α-GFP), infected with VSVG, and subjected to the 40°C block. The cells were then fixed 30 min after the 40°C block release, to allow VSVG to reach the Golgi complex, and processed for immuno-gold EM with an anti-GFP ab, to reveal cPLA₂α-GFP localization. After activation of VSVG transport, a cPLA₂α-GFP signal was detected at the rims of the Golgi cisternae and flanking tubular structures (arrows). (I, J) Quantification of gold labelling (mean±SD; n = 30 stacks) at the Golgi complex (see Materials and Methods) shows most of the cPLA₂α-GFP is bound to the tubular profiles (I) and the rims of the cisternae (J). (K, L) HeLa cells were co-transfected with the cDNAs encoding VSVG-YFP and the C2 domain of cPLA₂α fused with GFP (C2-GFP), kept at 40°C for 3 h (K), and observed in vivo under the confocal microscope during VSVG-YFP release from the ER. Images extracted from the time-lapse sequence show that C2-GFP moves from the cytosol (K) to the Golgi membranes (L) when VSVG appears within the Golgi area. (M) HeLa cells were transfected and incubated as above (K, L), and then observed under the confocal microscope and fixed during VSVG release when detectable amounts of C2-GFP started to appear in the perinuclear area. Further staining with anti-giantin antibodies reveals overlap of C2-GFP perinuclear signal with giantin labelling (arrows). Scale bar, 7 µm (A–D, F, G), 160 nm (H), 9 µm (K–M).

Control (A, B) and cPLA₂α-silenced (C) HeLa cells were prepared for EM tomography after chemical fixation. (A, B) The digital slice (A) and zoom image (B; corresponding to the area outlined by black box in A) extracted from the tomogram (see Video S2) shows an intercisternal connection (arrow) bridging cisternae located at the different levels of the stack in control cells. (C) Digital image extracted from the tomogram reveals no bridges between cisternae within the same Golgi stack (see Video S3) as well as a lack of connection between neighbouring stacks (indicated by arrowheads). (D) Quantification of vertical connections per stack in sections (mean±s.e.; n = 10 stacks) in EM tomograms (see Materials and Methods), in control and cPLA₂α-siRNAs-treated HeLa cells. Scale bar, 150 nm (A, C), 50 nm (B).

(A–F) HFs were infected with VSV and kept at 40°C for 3 h to accumulate VSVG in the ER. The cells were microinjected with an anti-cPLA₂α ab (mixed with FITC dextran) during the block, then shifted to the permissive temperature (32°C) and fixed at different times. (A, B) Staining of non-permeabilized cells with an ab against the ectodomain of VSVG 60 min after the 40°C block release shows reduced VSVG at the cell surface in microinjected cells (asterisks). (C, D) Microinjection with an anti-cPLA₂α ab (asterisks) induces fragmentation of the Golgi complex, prevents delivery of VSVG to the plasma membrane, and blocks VSVG within the early Golgi compartment without showing overlap with TGN46 (compare insets 1 and 2 in panel D, which correspond to dashed boxes in the control and injected cells). (E, F) Morphometric quantification shows poor colocalization of TGN46 with VSVG (E; mean±SD; n = 50 cells) and a reduction in the number of post-Golgi carriers (F; mean±SD; n = 50 cells) in anti-cPLA₂α-ab-microinjected cells, compared to mock-injected cells. Scale bar, 30 µm (A, B), 7.5 µm (C, D).

(A, B) HeLa cells were co-transfected with cPLA₂α-GFP and VSVG-Cherry and exposed to the 40°C block to accumulate VSVG-Cherry within the ER. Pyrrolidine (1 µM; Pyr) was added to the cells (B) 15 min before block release. Then cells were shifted to 32°C in the absence (A) or in the presence (B) of pyrrolidine for 45 min, fixed, and investigated under confocal microscopy. In control cells, VSVG-Cherry was detected at the cell surface (A) and its delivery to the PM was inhibited in pyrrolidine-treated cells (B) ,while cPLA₂α-GFP recruitment to the Golgi membranes was not affected by inhibitor treatment (compare A and B). (C–G) Control (C) and cPLA₂α-silenced (D–G) mouse MC3T3 cells were transfected with VSVG-Cherry alone (C, D) or in combination with the following human cPLA₂α constructs: cPLA₂α-GFP (E), cPLA₂α^(1–522) (F), cPLA₂α^S228C (G). The cells were subjected to 40°C block, then shifted to 32°C (to activate VSVG-Cherry exit from the ER) for 45 min, fixed, and stained with an anti-cPLA₂α ab. Confocal microscopy revealed efficient delivery of VSVG-Cherry to the PM in control cells (C), while in silenced cells, most of the VSVG-Cherry remained within the fragmented Golgi complex (D). Transfection of wild-type cPLA₂α-GFP rescued VSVG-Cherry delivery to the cell surface in cPLA₂α-silenced cells, while expression of the catalytically inactive mutants cPLA₂α^(1–522) (F) or cPLA₂α^S228C (G) did not reactivate VSVG-Cherry transport. (H) PCCL3 cells were loaded with [³H]-AA (see Materials and Methods). Part of the loaded cells was infected with VSV. Then infected and noninfected (control) cells were exposed to the 40°C block, washed, and shifted to 32°C. Fresh medium was added to the cells for 3 min time intervals and then collected. The [³H]-AA released into the medium over each 3 min-long interval was measured (see Materials and Methods). Quantification of the AA release (mean±SD; n = 6 experiments) revealed increase of PLA₂ activity (with the peak at 15 min) in VSV infected cells as compared to control. (I–L) HeLa cells were loaded with bis-BODIPY FL C₁₁-PC (see Materials and Methods) and fixed directly (I) or 15 min after incubation with either 5 µM ionomycin (J) or 1 µM pyrrolidine (K). The fluorescent signal indicating PLA₂ activity was detected by confocal microscopy both in the perinuclear region and the periphery of control cells (I). This signal increased after ionomycin treatment (J), but decreased after pyrrolidine addition (K), as also revealed by quantification (L) of the mean fluorescence per cell (mean±SD; n = 20 cells). (M, O) HeLa cells were infected with VSV, kept at 40°C for 3 h, and loaded with bis-BODIPY FL C₁₁-PC at the end of 40°C block. The cells were then fixed directly at the end of the 40°C incubation (M) or at different time intervals after temperature shift to 32°C (N, O). The cells were then stained with antibodies against VSVG and GM130 (Golgi marker). Confocal microscopy revealed only moderate PLA₂ activity overlapping with the Golgi when VSVG resided within the ER (M) while activation of VSVG transport through the Golgi (15 min after release from the ER) induced an increase in the BODIPY signal in the Golgi area (N). Quantification of the mean BODIPY fluorescence per cell (mean±SD; n = 20 cells) indicates an increase in PLA₂ activity in cells actively transporting VSVG, as compared to uninfected (control) cells (O). Scale bar, 8.5 µm (A–G), 16 µm (I–K), 7 µm (M, N).

HeLa cells were incubated with control siRNAs or siRNAs specific for cPLA₂α, GVIIIA-PLA₂, or both for 72 h. (A) Cells exposed to these siRNAs were infected with VSV and kept for 6 h at 32°C to allow continuous synthesis and transport of VSVG through the secretory pathway (“steady-state” conditions). Then the cells were fixed and VSVG at the cell surface was stained with an ab against its ectodomain. Afterwards, the cells were permeabilized and incubated again with an anti-VSVG ab to reveal the total pool of VSVG within the cell. Scale bar, 10 µm. (B) Cells were treated as in A or subjected to the 40°C block and then shifted to the permissive temperature of 32°C for 60 min (“traffic wave” conditions), fixed, and labelled for surface and total VSVG as described above. Then the fluorescence intensities of surface and total VSVG were evaluated, expressed as a ratio, and normalized to the control. This quantification reveals that under both “steady-state” and “traffic-wave” conditions, cPLA₂α silencing inhibits VSVG transport much more strongly than GVIIIA-PLA₂ depletion (see also images in panel A). RNAi of both PLA₂ enzymes, in turn, shows a slightly stronger inhibitory effect on VSVG transport over cPLA₂α silencing alone. (C) Cells incubated with different siRNAs (as described above) were subjected to western blotting with antibodies against either cPLA₂α or GVIIIA-PLA₂ or GAPDH, as indicated.