Misteli, Thomas A.; Studies on the mitotic disassembly of the Golgi apparatus in mammalian cells. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

During interphase, the Golgi apparatus of mammalian cells exists as a reticulum of interconnected slacks, each made up of several flattened, closely apposed cisternae. As cells enter M-phase, the Golgi apparatus disassembles and its membrane is found in clusters of small vesicles and short tubules by pro-metaphase. In order to study the steps leading to this reorganisation, a morphological cell-free system was developed and characterised. Isolated Golgi membranes were incubated with cytosol from mitotically arrested cells and intermediates of the disassembly pathway were identified by qualitative electron microscopy. The sequence of steps leading to complete fragmentation of stacks in the cell-free system was characterised by stereological analysis. Stacks fragmented by continued shedding of membrane from the periphery, before unstacking and complete fragmentation into a population of small vesicles and short tubules. COP-coated vesicles, implied in intra-Golgi transport, appeared to contribute substantially to the removal of membrane from the periphery of cisternae. The early steps of COP-coated vesicle biogenesis, budding and uncoating, were not affected by mitotic conditions, whereas their fusion with the target membrane appeared inhibited. The formation of COP-coated vesicles under mitotic conditions could not account for the complete fragmentation of the stack, and evidence for an alternative mechanism of fragmentation was found. For biochemical analysis, a quantitative assay based on the altered sedimentation behaviour of fragmented membranes was developed. This assay was used to investigate the regulation of fragmentation by a phos-phatase-kinase cycle, and the direct regulation of the fragmentation process by the mitotic master regulator cdc2-kinase was demonstrated. Finally, a temperature-sensitive mammalian mutant cell-line, defective in the cdc2-kinase gene, was used to characterise the disassembly of the Golgi apparatus at the electron microscopy level in-vivo. The in-vivo findings largely confirmed the in-vitro results.

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