Partitioning of the Golgi apparatus during mitosis in living HeLa cells.

Abstract

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I-GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.