Assembly of an intact Golgi complex requires phospholipase A2 (PLA2) activity, membrane tubules, and dynein-mediated microtubule transport

Previous studies have shown that treatment of mammalian cells with phospholipase A2 (PLA2) antagonists cause the normally interconnected Golgi ribbon to break up into large fragments of stacked Golgi cisternae (“mini-stacks”) that remain located in the juxtanuclear region. Using the reversible PLA2 antagonist, ONO-RS-082 (ONO) and live-cell, time-lapse microscopy to image the Golgi reassembly process, we found that Golgi mini-stacks underwent a burst of membrane tubule formation following washout of ONO: before washout only 4.3 ± 3.8 tubules/cell/10 min were formed, whereas after washout 29.9 ± 11.9 tubules/cell/10 min formed. These membranes tubules formed bridges between physically separate mini-stacks, thus mediating their coalescence into intact Golgi ribbons. Formation of inter-stack tubules and an intact Golgi ribbon was also facilitated by microtubules because treatment with nocodazole significantly inhibited both processes. This microtubule-dependent process was also dependent on dynein because the dynein inhibitor nordihydroguaiaretic acid (NDGA) inhibited reassembly. These studies show that a late stage of Golgi assembly occurs via membrane tubules, whose formation is dependent on PLA2 activity and microtubules. Considering these results together, we concluded that the maintenance and assembly of normal Golgi architecture is dependent on the PLA2-mediated, dynamic formation of inter-Golgi membrane tubules.