A role for phospholipase A2 activity in membrane tubule formation and TGN trafficking

Abstract

We have investigated the role of phospholipase A(2) (PLA(2) ) enzymes in generating membrane tubules at the trans-Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over-expressing kinase dead protein kinase D were inhibited by the PLA(2) inhibitors ONO-RS-082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time-lapse imaging revealed for the first time that PLA(2) antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA(2) enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking.

Figure 1. PLA₂ antagonists inhibit PKD-KD-induced TGN membrane tubules

HeLa cells transfected with PKD-KD-GFP were incubated with 10 μm ONO or a solvent control for up to 60 min. A) Cells without ONO had abundant TGN membrane tubules containing PKD-KD-GFP while after treatment with ONO for 30 min, few cells contained tubules. B) Cells were counted as containing TGN membrane tubules, or not, for each time-point. C) HeLa cells transfected with PKD-KD-GFP were treated with varying concentrations of ONO for 30 min. The number of cells with TGN tubules was counted. D) HeLa cells transfected with PKD-KD-GFP were treated with 10 μm BEL for up to 90 min. The number of cells containing TGN membrane tubules was counted. Error bars represent standard deviation for three independent experiments and 300 cells counted. Scale = 10 μm.

Figure 2. PKD-KD-GFP-induced TGN membrane tubules contain proteins destined for the plasma membrane and endosomes

HeLa cells transfected with PKD-KD-GFP were incubated at 20°C for 2 h to block TGN secretion. Cells were shifted to the permissive temperature of 32°C for 30 min and then fixed and stained with antibodies against various TGN cargo proteins. Line intensity plots were generated using NIH ImageJ software. Scale = 10 μm.

Figure 3. PLA₂ antagonists inhibit the formation of TGN membrane tubules

HeLa cells transfected with PKD-KD-GFP were treated with 10 μm ONO for 60 min. The media was then replaced without ONO and cells were visualized at various periods of time thereafter. A) Within 15 min of ONO washout, the TGN reformed membrane tubules which became more numerous and extensive after 30 and 60 min. B) Cells were counted for containing TGN membrane tubules at various time-points after ONO washout. After 60 min, nearly all cells contained tubules. Error bars represent standard deviation for three independent experiments and 300 cells counted. Scale = 10 μm.

Figure 4. Live-cell imaging of membrane tubules in cells over-expressing PKD-KD-GFP or ts045VSV-G-YFP

In untreated cells, membrane tubules are dynamic in that they form, retract and/or break off. In cells treated with 10 μm ONO, membrane tubules similarly retract and break off, but do not reform. A) Control and ONO treated (10 μm) cells expressing PKD-KD-GFP. Micrographs were taken from Movies S1 and S2. B) Control and ONO treated (10 μm) cells expressing ts045VSV-G-YFP. Micrographs were taken from Movies S3 and S4. Red arrows indicate forming membrane tubules while yellow triangles indicate retracting tubules. C) Quantification of ts045VSV-G-YFP membrane tubules formed from the TGN over 5 min. Data represents three independent experiments with over 30 cells counted. A two-tailed Student's t-test with unequal variance was performed. A) Scale = 5 μm. B) Scale = 10 μm.

Figure 5. Constitutive trafficking from the TGN to the plasma membrane is inhibited by PLA₂ antagonists

HeLa cells were transfected with empty vector or PKD-KD-GFP for 24 h followed by transfection with ssHRP-FLAG for 24 h. Cells media was replaced and media samples were taken at the indicated times. A) Cells treated with 10 μm ONO had complete inhibition of ssHRP secretion and cells over-expressing PKD-KD-GFP had approximately 50% reduction in secretion. B) Cells transfected with ts045VSV-G-DsRed were incubated at 40°C for 18 h then 20°C for 3 h and then permissive temperature of 32°C for 60 min. Cells without ONO readily transported VSV-G to the plasma membrane within 60 min at the permissive temperature, while cells treated with 10 μm ONO retained VSV-G in the TGN. C) TGN-localized VSV-G was quantified by measuring fluorescence intensity of DsRed in the TGN versus the entire cell. Numbers were normalized to control and show that ONO blocked ts045VSV-G-DsRed escape from the TGN. Scale = 10 μm.