The Golgi apparatus has been fluorescently tagged using GFP localized on the lumenal side of the membrane by the medial
retention signal of NAGT I. The efficacy of this tag was confirmed both by fluorescence and immunoelectron microscopy. Immunogold labeling showed that the hybrid protein was mostly restricted to one or two cisternae on one side of the stack. Confocal microscopy showed that NAGFP was sandwiched between the cis
Golgi markers GM130 or p115, and the TGN marker, TGN46. It was often surrounded by the peripheral protein, giantin, and most importantly, colocalized most completely with Mann II. Since Mann II has the same location as NAGT I in HeLa cells (Rabouille et al., 1995
), these results strongly suggest that NAGFP is present in the same location as the parent NAGT I, namely the medial
cisternae. As such, it provides an excellent vital marker for the Golgi apparatus.
Though transient transfection with the NAGFP cDNA resulted in high levels of Golgi fluorescence in HeLa, NRK, and embryonic stem cells (unpublished observation), significant levels of fluorescence in stable HeLa cell lines were only obtained after treatment with butyrate. A 16-h incubation increased synthesis of the protein by about fivefold, and this was accompanied by a dramatic increase in the level of fluorescence. Treatment with butyrate for less than 24 h had been shown by others to have no effect on cell cycle progression in HeLa cells (Darnell, 1984
). This was confirmed for the NAGFP-HeLa cells, which were also shown to be viable and fluorescent for several days after stimulation. This permitted studies of the Golgi apparatus during the cell cycle. The difficulty in obtaining cell lines stably expressing fluorescent GFP has been experienced by others (Cubitt et al., 1995
; Olson et al., 1995
) and suggests that continuous expression can be toxic. Time could well be saved if initial attempts focused on inducible systems, using either specific promoters or less specific treatments, such as butyrate.
Fluorescent tagging of the Golgi apparatus using GFP has permitted, for the first time, continuous examination of the Golgi apparatus in mitotic cells. Intermediates involved in the partitioning of mitotic Golgi membranes were identified and followed as cells progressed through mitosis. The first important feature to emerge from these studies is that metaphase cells contained a constant number (130 ± 2, n = 5) of dispersed mitotic Golgi fragments, shown by EM to be tubulo-vesicular mitotic clusters. This finding strongly suggests that the end product of Golgi breakdown is not the vesicle but the Golgi cluster.
Earlier EM studies had not resolved this issue. Quantitation of Golgi cluster profiles impregnated with osmium showed that there were ~140 mitotic clusters/metaphase cell, similar to the number now found using NAGFP (Lucocq and Warren, 1987
). Cryo-immuno–EM, however, showed that clusters could shed vesicles into the surrounding cytoplasm, raising the possibility that shedding of vesicles could go to completion, making the vesicle the end product (Lucocq et al., 1989
). Immunofluorescence analysis provided no firmer data, in part because of the lack of availability of high-titre, high-affinity antibodies and because of technical difficulties due to high background fluorescence and abundant out-of-focus material in rounded, mitotic cells.
The GFP has helped resolve this problem, providing an invaluable tool for the investigation of subcellular dynamics and organization. In addition to the ability to follow living cells, the GFP tag also provides a level of sensitivity in fixed-cell fluorescence microscopy that cannot be readily achieved with conventional antibody-based methods. The virtual absence of background noise, coupled with the intense fluorescence signal obtained with this particular GFP variant (Cormack et al., 1996
), have enabled the investigation of subcellular membranes whose sizes are approaching the limits of resolution for the light microscope. This gain in sensitivity becomes even more significant when examining mitotic cells, whose intracellular structures are difficult to visualize because of the high degree of light scatter and increase in sample thickness (Cheng and Kriete, 1995
). The benefits of the increase in sensitivity and resolution are best demonstrated by the highly reproducible quantitation of metaphase cluster number in confocal fluorescence images (see above and Fig. ). Moreover, as shown in the multiple-label analysis of Golgi polarity, the endogenous fluorescent properties of the polarized NAGFP provide new opportunities for the analysis of Golgi resident organization. Paired with confocal microscopy, the NAGFP tag provides a rapid and simple means to obtain a whole cell overview of Golgi membrane distribution and organization.
A second feature to emerge from these studies is that mitotic clusters contained representatives of all resident markers tested (with the exception of p115; see below), and unexpectedly, these markers had the same distribution, relative to each other, as in the interphase Golgi apparatus. In other words, Golgi residents within the mitotic clusters were polarized. It could be argued that the mitotic NAGFP-HeLa cells represent a unique case. Several experiments were performed to address this concern. First, the transformation of Golgi stacks into mitotic clusters was confirmed by quantitating Golgi profiles in electron micrographs. All Golgi profiles found in metaphase cells were mitotic clusters. At the EM level, these clusters were indistinguishable from those described previously for mitotic cells in the parotid gland (Tamaki and Yamashina, 1991
), thyroid epithelium (Zeligs and Wollman, 1979
), and the parental HeLa cell line (Lucocq et al., 1987
); therefore, by morphological criteria, Golgi membranes in the metaphase NAGFP-HeLa are indistinguishable from those observed during previous studies of mitotic Golgi membranes. In addition, this analysis excludes the possibility that the polarized metaphase Golgi fragments in the NAGFP-HeLa cells are just stacks that have failed to convert into clusters. Second, mitotic clusters were present in NAGFP-HeLa cells that have not been treated with butyrate or aphidicolin, parental HeLa cells, and primary human keratinocytes during all stages of mitosis (Fig. and unpublished observations). This provides strong evidence that mitotic clusters are not an artifact of drug treatments or GFP expression and are not restricted to tumor cell lines, but are a common unit of Golgi partitioning. Lastly, mitotic Golgi membranes in the parental HeLa cell line and primary human keratinocytes were polarized; thus, we conclude that the formation of polarized mitotic clusters is a common element of the Golgi fragmentation process during cell division.
The failure to observe cluster polarity in the past is likely due to the predominantly ultrastructural nature of mitotic Golgi analysis; it was assumed that the loss of cisternal morphology in mitotic Golgi membranes resulted in the loss of biochemical polarity. Furthermore, the loss of cisternal morphology makes it difficult to determine the relative orientation of resident Golgi proteins within the cluster unless cryo-immunolabeled sections (~60–80-nm thickness) are reconstructed into three-dimensional, double-label images of the cluster (~500-nm diameter). This is currently not feasible. The analysis of mitotic Golgi by confocal microscopy has avoided this problem since the volume of illumination is approximately the same size or slightly larger than the mitotic clusters. This feature has permitted the visualization of entire clusters within a single optical section and greatly enhances the ability to observe the compartmental nature of Golgi protein distribution.
The maintenance of polarity in the apparently disorganized membranes of the cluster may be explained by the COP I–independent disassembly of the resident-enriched core regions of the Golgi stack. In contrast to the COP I pathway, which appears to convert the transport-specialized cisternal rims into coated vesicles, the COP I–independent pathway results in an increase in Golgi membrane fenestrations and the formation of extensive tubular networks (Misteli and Warren, 1995b
). In thin section electron micrographs, tubular–reticular networks would appear as a heterogeneous collection of vesicles and tubules, similar to the morphology of Golgi membranes in the cluster. Hence, we speculate that during mitosis, extensive tubulation of core Golgi cisternae in situ leads to the transformation of cisternae into tubulo-vesicular membranes without disrupting their distribution relative to other cisternae in the stack.
The polarized distribution of resident proteins in the Golgi clusters suggests the existence of a structural template onto which the Golgi stack is reorganized after mitosis. The membranes themselves may act as the structural template, harboring the information sufficient for self- association and organization of the dispersed Golgi components: perhaps Golgi “adhesion molecules” are responsible for maintaining cluster compartmentation, similar to the proposed role for cell–cell adhesion molecules (e.g., cadherins) in sorting out cell populations into patterned tissues (Steinberg, 1963
). Alternatively, mitotic membranes might be organized upon an underlying scaffold that determines Golgi architecture. Cytoskeletal-like Golgi proteins, such as giantin, Golgi spectrin, or GM130, could participate in the construction of such a structure (Barr and Warren, 1996
The vesicle docking protein p115 is the only Golgi marker so far tested that was absent from the mitotic clusters. We have previously shown that p115 binds with a 10- to 20-fold lower affinity to Golgi membranes after treatment with mitotic cytosol (Levine et al., 1996
). The present work provides strong evidence that this also occurs in vivo and supports our hypothesis that the COP I–dependent fragmentation of the Golgi apparatus results from an inability of the COP I vesicles to dock and therefore fuse with their target membranes. In contrast to mitotic clusters, p115 is found on the dispersed Golgi stacks generated by treatment with nocodazole (Fig. ). This is consistent with the fact that these stacks carry out exocytic transport (Featherstone et al., 1985
; Cole et al., 1996a
) and provides a means of distinguishing these small Golgi stacks from mitotic clusters.
The persistence of a constant number of mitotic clusters does not preclude the presence of free vesicles or tubules derived from them. Their size and dilution would hinder detection by current fluorescence techniques. In addition, the products of the COP I–dependent pathway are vesicles depleted in the resident proteins used as markers for the Golgi compartment. The only evidence for loss of membrane from clusters comes from measurements of apparent cluster size. These range from 0.2 to 0.9 μm and suggest that some clusters could lose more membrane (in the form of vesicles) than others. However, shedding of vesicles into the cytoplasm does not go to completion; therefore, clusters are not intermediates on the fragmentation pathway, they are end products.
The accuracy of partitioning will be limited by the Golgi unit present in the least copy number. Such a unit must also be able to seed the regrowth of the complete organelle once mitosis is complete. By these criteria, the mitotic clusters are the unit of Golgi partitioning. They have representatives of all the biochemical compartments of the Golgi, and they are present in the lowest copy number. Shed components and molecules such as p115 are not likely to seed regrowth, and their greater number (and smaller size) means that they would be at least as accurately partitioned as the limiting clusters.
In living cells, individual metaphase clusters displayed no obvious directed movement that could be categorized. Instead, as cells divided, the cluster population appeared to move as a collective in the direction of the spindle poles, suggesting that the clusters may be anchored to an underlying structure such as aster microtubules or the mitotic spindle (Lucocq et al., 1989
). In late telophase/G1, the Golgi membranes congregated in the presumed pericentriolar region and, slowly, by an iterative process, reformed the interphase ribbon. Two types of directed movement were noted during this period: condensation of telophase Golgi membranes to form 1–3-μm fragments and extension of tubules to join the fragments into a rudimentary Golgi ribbon. Both cytoplasmic dynein and kinesin have previously been implicated in the directed movement of Golgi membranes along microtubules. Cytoplasmic dynein is necessary for the cell-free movement of Golgi fragments towards the centrosome (Corthesy-Theulaz et al., 1992
), and brefeldin A–stimulated formation of ER-directed tubules is kinesin dependent (Lippincott-Schwartz et al., 1995
). The ability to track Golgi membranes in living, dividing cells should provide a powerful system for identifying the motor components responsible for rebuilding the interphase Golgi ribbon in vivo.
The orderly and directed breakdown, distribution, and reformation of the Golgi apparatus implies that to facilitate the partitioning of the Golgi, a mechanism more sophisticated than one that simply randomizes the Golgi membranes is required. This raises the question of whether the distribution of Golgi clusters in the metaphase cell serves to increase the accuracy of a partitioning process that relies solely on cytokinesis, or if it reflects a more ordered mechanism of sorting Golgi membranes into daughter cells. Ordered mechanisms for partitioning multicopy organelles were first proposed in the early part of this century when the partitioning of mitochondria during spermatogenesis was shown to be more accurate than that predicted by a simple stochastic mechanism (Wilson, 1916
; Birky, 1983
). Attempts to analyze the accuracy of Golgi partitioning by EM were originally carried out using osmication to identify Golgi membranes in thick sections. This permitted the number of clusters in each daughter cell to be determined, but the accuracy of the values obtained was limited by the small sample size since the technique required complete serial sectioning of each dividing cell (Lucocq and Warren, 1987
). The NAGFP-HeLa cells have provided a more convenient system for determining the accuracy of partitioning. Measurements based on the partitioning of the GFP tag show that the experimentally determined accuracy was ~2.5-fold better than would be predicted for a stochastic event. These findings provide evidence that the mitotic partitioning of the Golgi occurs through an ordered mechanism; however, definitive proof awaits the characterization of the putative mechanism(s).
Regardless of the nature of the partitioning mechanism, the constant number of clusters in metaphase cells points to a biosynthetic mechanism that accurately maintains this number. The mechanism is unknown, but the level of Golgi membrane in a cell appears to reflect the amount of plasma membrane that it must service. For example, Xenopus
oocytes have a low surface area to volume ratio and contain comparatively little Golgi membrane (Colman et al., 1985
). Moreover, we found that mitotic cells synchronized with aphidicolin were larger (and therefore had more plasma membrane) than those in unsynchronized populations (compare cell sizes in Figs. a
and 10 a
). They had a larger (though still constant) number of similarly sized metaphase clusters (130 vs. ~70). This raises the possibility of a relationship between the area of plasma membrane, volume of the Golgi apparatus, and mitotic cluster number.
The mechanism for maintaining a constant number of Golgi units could operate in one of two ways: either it could rely on the synthesis of new copies of the Golgi apparatus de novo, or it could control the growth and division of preexisting Golgi membranes. The present work has raised the possibility that an underlying Golgi scaffold persists throughout the cell cycle, even when the cisternal membranes themselves are disassembled. If new copies of the Golgi arise de novo, this scaffold would be capable of self-assembly, forming a limiting structure of defined size. Alternatively, the scaffold could act as a template on which another copy would be built. Examination of NAGFPHeLa cells during the period of Golgi biogenesis should provide insight into which of these two mechanisms operates.
Disassembly of the Golgi apparatus into dispersed vesicles and tubules was thought to increase the accuracy of a stochastic partitioning process. The present work, however, suggests a more ordered partitioning mechanism, which raises the question: why locally fragment the stacks into tubulo-vesicular clusters? In plants and fungi, the Golgi exists as discrete, dispersed stacks throughout the cell cycle, suggesting that the stack can function as an effective unit of partitioning (for review see Warren, 1993
). The retention of a stacked morphology in plants and fungi could reflect the need for continuous secretion to synthesize new cell wall material during mitosis. In contrast, in animal cells, there is a general cessation of membrane traffic during mitosis (Warren, 1985
), so there may not be a requirement to maintain the stacked morphology. Instead, the inhibition of secretory traffic at mitosis might contribute to the local disassembly of stacks into membrane clusters. However, even in this speculative scenario, the functional significance of cluster formation is not obvious.
One possible function of cluster formation is to increase access to an area of membrane equivalent in surface area to that of the plasma membrane (Griffiths et al., 1984
). Examination of clusters by EM shows that cluster membranes are separated from each other, much more so than when they are present in stacked cisternae (Misteli and Warren, 1995a
). Such a process could either expose membrane-bound proteins that are stored in the stacked regions during interphase but required during mitosis, or sequester interphase components that might otherwise hinder the mitotic process. A number of signaling molecules have been localized to Golgi membranes, including phospholipase D (Ktistakis et al., 1995
) and various isoforms of protein kinase C (Goodnight et al., 1995
; Lehel et al., 1995
). In addition, proteins that regulate the distribution of their associated kinases during interphase and mitosis are also present on the Golgi. These include the regulatory subunit of protein kinase A (Nigg et al., 1985
) and cyclin B2 (Jackman et al., 1995
In summary, we have devised a means of following the fate of the Golgi apparatus in living cells during the mammalian cell cycle. The system has provided insight into Golgi biogenesis and partitioning and will be of use in investigating various aspects of organelle function during the cellular growth and division process.