Peat, Deborah Nicole; (1993) Fractionation of Golgi membranes from mitotic HeLa cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

During mitosis the cisternae of the mammalian Golgi apparatus are fragmented into thousands of vesicles. To study whether this change in morphology is reflected in the physical properties of Golgi membranes, the fractionation of mannosidase-l, a marker for the cis Golgi cisternae, and β-1,4-galactosyltransferase a trans cisternae Golgi marker was analysed on sucrose gradients. A method was developed for the growth of large numbers of HeLa cells in suspension culture which were subsequently blocked in prometaphase with the microtubule inhibitor nocodazole for 24 hours for the production of mitotic cells. The morphology and biochemistry of these cells was very similar to that previously described for mitotic cells isolated from roller bottles by shake-off. A reproducible protocol for the homogenisation of mitotic cells was developed and used for the fractionation of organelles by equilibrium density centrifugation in sucrose. This study shows: 1. The Golgi apparatus membrane marker β-1,4-galactosyltransferase of both interphase and mitotic HeLa cells has an equilibrium density of 1.129g/cm3. 2. The cisternal membranes of the Golgi apparatus retain their biochemical integrity and remain discrete during mitosis, neither mixing with each other, nor with markers for any other organelle. 3. A fundamental difference in the equilibrium density of the two resident Golgi enzymes mannosidase-l and β-1,4-galactosyltransferase in mitotic cells. The cis membrane marker undergoes a dramatic and reversible shift to a lower equilibrium density during mitosis while the trans membrane marker density remains unchanged. The density shift is consistent with the loss of peripheral cytoplasmic protein and similar to the shift described for clathrin-coated endosomal vesicles (Rothman and Schmid, 1985; Woodman and Warren, 1991). 4. Purification of membrane vesicles enriched in β-1,4-galactosyl- transferase activity from mitotic HeLa cells gave a 20-fold purification with 3% yield when sucrose gradient centrifugation was followed by further fractionation on Percoll gradients. Vesicles were examined by electron microscopy and shown to be morphologically similar to the mitotic Golgi vesicles described by Lucocq et al., 1987 for intact mitotic cells.

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