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Mini-Review |
Address correspondence to Francis Barr, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, Martinsried, 82152 Germany. Tel.: 49-89-8578-3135. Fax: 49-89-8578-3102. email: barr{at}biochem.mpg.de
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Abstract |
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Key Words: mitosis; Golgi fragmentation; Golgi haze; FRAP; rapamycin
What is the fate of the mammalian Golgi apparatus during mitosis? This seemingly simple question has become part of the wider debate concerning the nature of the Golgi apparatus and the mechanism of secretory protein transport. At the heart of this debate is the question of whether the Golgi is an organelle with its own separate identity, or a complex transport intermediate containing secretory cargo that is populated by enzymes rapidly recycling to and from the ER (Glick, 2002). In recent years, this debate has been propelled by the use of microscopy techniques that allow fluorescent protein tagged Golgi enzymes to be detected in living cells, and quantitative measurements of their localization, rates of transport between the ER and Golgi, and their diffusion rates in these two compartments to be made (Cole et al., 1996). The initially surprising results from the use of such techniques were that Golgi enzymes showed high diffusional mobility and are recycling between the ER and Golgi, so that at steady state over 30% of ß1, 4-galactosyltransferase, a medial/trans-Golgi enzyme, was present in the ER (Cole et al., 1996; Zaal et al., 1999). Meanwhile, other observations showed that imposing a block on the COPII vesicle formation pathway used by cargo molecules exiting the ER by introducing dominant-negative forms of the Sar1 GTPase into cells lead to the redistribution of Golgi enzymes back into the ER (Storrie et al., 1998). This is similar to the phenotype of brefeldin A (BFA)treated cells, where the Golgi fuses with the ER due to deregulation of the ARF1 GTPase and its associated coat proteins (Lippincott-Schwartz et al., 1989). These findings lead to the proposal that Golgi proteins might accumulate in the ER during mitosis as a result of their normal interphase recycling pathway (Fig. 1), combined with the mitotic block in protein transport between the ER and Golgi (Zaal et al., 1999; Lippincott-Schwartz and Zaal, 2000). Older observations had also suggested that such a pathway might exist in mitotically arrested cells where Golgi enzymes were found in the ER (Thyberg and Moskalewski, 1992). This was an alternative to a previous model (Fig. 1) in which the Golgi was said to directly fragment into many small vesicles and tubular remnants (Lucocq et al., 1987; Shima et al., 1997). Various lines of evidence were provided to support the conclusion that in mitosis Golgi enzymes and lipids were present within a large continuous membrane system, namely the ER, rather than in unconnected fragments derived from the Golgi (Zaal et al., 1999). These findings have now been reinvestigated by two groups taking complementary approaches for determining the fate of the Golgi in mitosis (Axelsson and Warren, 2004; Pecot and Malhotra, 2004).
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Trapping ER and Golgi structures in mitosis |
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These findings support previous biochemical studies from a number of groups, where markers of the ER and Golgi could be separated using cell fractionation techniques (Jesch and Linstedt, 1998; Jesch et al., 2001; Seemann et al., 2002). The rapamycin trapping-assay complements these cell fractionation approaches because it shows that the compartment containing Golgi enzymes in mitotic cells is unlikely to be generated via a recycling pathway involving the ER. One caveat to this is the assumption that Golgi contents should become sufficiently mixed with the ER during recycling to allow trapping to occur, which is not necessarily the case if recycling occurs at subdomains of the ER from which ER-retained proteins are excluded and to which proteins recycling to the Golgi are restricted. However, this seems unlikely given the high diffusional mobility observed for proteins within the ER, and measurements suggesting they have access to the entire ER volume (Ellenberg et al., 1997).
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Seeing through the mitotic Golgi haze |
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Clarifying the mechanism of Golgi inheritance |
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Why do the vesicles making up the Golgi haze diffuse so rapidly in mitotic cells, much more rapidly than fragmented ER vesicles? The cytoplasm is crowded with many large protein assemblies and organelles, and this would be expected to have an effect on vesicle diffusion, albeit one that is not necessarily predictable (Seksek et al., 1997; Verkman, 2002). However, at present it is unknown how the properties of the cytoplasm alter measurements of vesicle diffusion, the precise effects of vesicle size, and what volume of the cytoplasm is actually available for vesicle diffusion in living cells and how this differs between interphase and mitosis. All of these points are important for a full interpretation of measurements made on membrane organelles such as the ER and Golgi.
There is also the question if fragmentation of the Golgi during mitosis has any function other than in organelle inheritance. One suggestion is that Golgi disassembly has a function in a form of organelle checkpoint regulating cell cycle progression at the G2/M phase transition (Sutterlin et al., 2002). However, this is an isolated observation and the mechanism is unknown. It is also worth noting that the studies from the Malhotra and Warren labs show that in BFA-treated cells where the Golgi is merged with the ER, progression through mitosis, cytokinesis, and Golgi inheritance are apparently normal (Axelsson and Warren, 2004; Pecot and Malhotra, 2004). The underlying reason, if any, for a Golgi fragmentation in mitosis separate from the ER is therefore still a mystery. One possibility is that the behavior of the Golgi in mitosis mirrors the different modes of cell division seen in fungi, plants, and vertebrate cells (Guertin et al., 2002). Evidence from studies of Drosophila melanogaster indicate that the fate of the Golgi mitosis can reflect differences in the growth state of the cells or organism. However, at the present time no real coherent picture has emerged and further studies will be needed to clarify this issue.
One point that all protagonists in this debate can surely agree on is that there are limitations to light microscopy, and there is a need for a time resolved electron microscopy and tomography study to simultaneously follow a variety of ER and Golgi markers through mitosis and definitively establish the fate of these organelles in mammalian cells.
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Acknowledgments |
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The Max-Planck Society supports research in the laboratory of F.A. Barr.
Submitted: 2 February 2004
Accepted: 17 February 2004
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References |
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