PARTITIONING OF THE GOLGI-APPARATUS DURING MITOSIS IN LIVING HELA-CELLS

The Golgi apparatus of HeLa cells was fluorescently tagged with a gree n fluorescent protein (GFP), localized by attachment to the NH2-termin al retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscop y using a variety of Golgi markers. The behavior of the fluorescent Go lgi marker was observed in fixed and living mitotic cells using confoc al microscopy. By metaphase, cells contained a constant number of Golg i fragments dispersed throughout the cytoplasm. Conventional and cryoi mmunoelectron microscopy showed that the NAGT I-GFP chimera (NAGFP)-po sitive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly lit tle effect on the polarity of Golgi membrane markers at the level of f luorescence microscopy. In living cells, there was little self-directe d movement of the clusters in the period from metaphase to early telop hase. In late telophase, the Golgi ribbon began to be reformed by a dy namic process of congregation and tubulation of the newly inherited Go lgi 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 o f partitioning and suggest that precise regulation of the number, posi tion, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.