Reconstitution of the Golgi reassembly process in semi-intact MDCK cells

The Golgi apparatus, which consists of stacks of cisternae during interphas e, is fragmented or dispersed throughout the cytoplasm at the onset of mito sis. A sea sponge metabolite, ilimaquinone (IQ), causes Golgi membranes to vesiculate. And after its removal, the vesiculated membranes reassemble int o stacks of cisternae in the perinuclear region. To study the mechanism of Golgi membrane dynamics during mitosis, we have reconstituted the reassembl y process of IQ-induced vesiculated Golgi membranes in streptolysin O-perme abilized Mardin-Darby canine kidney (MDCK) cells. Monitoring the dynamics o f Golgi membranes labeled with a green fluorescence protein (GFP)-tagged pr otein, we dissected the process into two elementary components: the reassem bly of vesiculated Golgi membranes into punctate structures; and the subseq uent reformation of these structures into stacks of cisternae near the nucl eus. Using morphometric analysis, we studied the kinetics and biochemical r equirements for the process, and revealed that an NEM-sensitive factor, cyt oplasmic dynein, and GTP binding protein were involved in the Golgi reassem bly. (C) 2000 Elsevier Science B.V. All rights reserved.