Previous studies have shown that treatment of mammalian cells with phospholipase A(2) (PLA(2)) antagonists cause the normally interconnected Golgi ribbon to break Lip 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 rnicrotubule-dependent process was also dependent oil 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 PLA(2) activity and microtubules. Considering these results together, we concluded that the maintenance and assembly of normal Golgi architecture is dependent on the PLA(2)-miediated, dynamic formation of inter-Golgi membrane tubules. (C) 2009 Elsevier Inc. All rights reserved.