Cell-cycle-specific Golgi fragmentation: how and why?

462

Cell-cycle-specific Golgi fragmentation: how and why? Opinion Antonino Colanzi, Christine Suetterlin and Vivek Malhotra The Golgi membranes, in the form of stacks of cisternae, are contained in the pericentriolar region of mammalian cells. During mitosis, these membranes are fragmented and dispersed throughout the cell. Recent studies are revealing the significance of pericentriolar position of the Golgi apparatus and mechanism by which these membranes are fragmented during mitosis. Addresses Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, USA  Correspondence: Vivek Malhotra; e-mail: [email protected]

Current Opinion in Cell Biology 2003, 15:462–467 This review comes from a themed issue on Membranes and organelles Edited by Alice Dautry-Varsat and Alberto Luini 0955-0674/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00067-X

Abbreviations ERK extracellular-signal-regulated kinase GRASP Golgi reassembly stacking protein MEK1 mitogen-activated protein kinase kinase 1 Plk1 polo-like kinase 1

Introduction Golgi membranes are involved in the post-translational modification, sorting and transport of proteins. The organization and intracellular location of Golgi membranes, however, varies among cell types. In Saccharomyces cerevisiae, these membranes as a few cisternae are randomly distributed in the cytoplasm [1]. In higher eukaryotes, Golgi membranes are organized into stacks of flattened cisternae, which are either distributed randomly in the cytoplasm (plant cells and early Drosophila embryos) or confined to the pericentriolar region (mammalian cells) [2–4]. The fate of Golgi membranes during mitosis is also cell-type-specific. Golgi membranes of yeast, plant cells and early Drosophila embryos do not undergo any obvious morphological change during budding (yeast), cell division (plants) or the nuclear duplication events (Drosophila embryos). Parasites such as Trichomonas vaginalis and T. gondii contain a single stack of Golgi cisternae, which grows in size before cell division; the stack is then sliced into two and each half given to the newly forming daughter cells [5,6]. Interestingly however, mammalian cells fragment their Golgi membranes during mitosis and the fragments are dispersed throughout the cytosol [4]. Current Opinion in Cell Biology 2003, 15:462–467

What is the evolutionary logic that makes Golgi localization and inheritance in mammalian cells so elaborate? We will discuss the answer to this question here.

The fate of Golgi membranes in mitosis: does the final form matter? More than two decades ago, fluorescence microscopy of mitotic cells revealed that Golgi membranes are fragmented and dispersed [7]. Since then, the general thinking about the fate of Golgi membranes in mitosis has undergone many ‘avatars’ (reincarnation), the form of which is investigator-dependent. On the one hand, Thyberg and Moskalewski [8] analyzed mitotic cells using immunoelectron microscopy and found that Golgi-resident proteins are relocated to the endoplasmic reticulum (ER). More recently, Lippincott-Schwartz and colleagues [9] have also reported that Golgi membranes are redistributed to the ER during mitosis. Warren and co-workers [10], on the other hand, have shown that Golgi membranes are fragmented into 1302 tubulo-reticular clusters and isolated vesicles. Linstedt and colleagues [11] favor the proposal that Golgi membranes in the form of small vesicles are distributed throughout the cytoplasm and are distinct from the ER. Hammond and Glick [12] find Golgi membranes diffusely dispersed in mitotic cells; however, this diffuse staining is not resolved into an ER form or small vesicles. Furthermore, Lucocq and colleagues [13] have reported that mitotic Golgi membranes are predominantly tubulo-reticular elements separated from the ER. They do, however, detect 20% of the Golgi proteins at the ER exit sites [13]. We have found that Golgi proteins are contained predominantly in tubuloreticular elements and separate from the ER [14]. So what is the final form of the Golgi membranes in mammalian mitotic cells, and how will we resolve this issue? Is it worth the effort? There is a general agreement amongst the ‘mitotic Golgi fragmentors’ that Golgi membranes undergo sequential disassembly during mitosis: In late prophase/pro-metaphase, the pericentriolar Golgi stacks break down into smaller pieces (mitotic Golgi blobs, a term coined by James Nelson of Stanford University [15]). Subsequently, between pro-metaphase and early anaphase Golgi membranes undergo further fragmentation and are found diffusely dispersed in the cytosol (known as the mitotic Golgi haze). The extent of Golgi proteins that appear as a haze varies, ranging from 30% in Ptk1 cells to 95% in HeLa cells [11,16]. The major issue in the field is whether the mitotic Golgi haze represents small and dispersed vesicles or Golgi proteins that are relocated into the ER. www.current-opinion.com

Cell-cycle-specific Golgi fragmentation Colanzi et al. 463

Subcellular fractionation of mitotic cells has revealed that Golgi membranes can be separated from the ER [11,17]. But it cannot be ruled out that these cells were enriched in early mitotic stages (prophase to early metaphase), when Golgi membranes are clearly separated from the ER. The argument presented by Lippincott-Schwartz and colleagues, that Golgi membranes are redistributed into the ER, is based on the following experiment [9]. Cells expressing GalTase–GFP (galactosyltransferase– green fluorescent protein) were allowed to progress into mitosis. A defined area of the cell was then photobleached. With time, the bleached area recovered fluorescence. The authors argue that the fluorescence recovery would occur in the given time window only if GalTase–GFP was contained in a contiguous membranous network. While this is a valid argument, it would be important to carry out the same procedure in cells where Golgi membranes are completely vesiculated but not fused with the ER, such as treatment of cells with ilimaquinone [18]. Under such conditions, the kinetics of fluorescence recovery would be different and would strengthen the proposal of Lippincott-Schwartz and colleagues that Golgi membranes are not vesiculated but fused with the ER in mitosis. An alternative procedure that could help resolve the composition of the mitotic Golgi haze is to employ correlative electron microscopy [19]. In this procedure, a cell that is specifically selected at the immunofluorescence level can be analyzed at the ultrastructural level to confirm the location of the Golgi

enzymes. Moreover, serial sections of this particular cell can help resolve the three-dimensional structure of the mitotic Golgi haze.

Mechanism of Golgi fragmentation As discussed above, the fragmentation of Golgi apparatus occurs in two sequential steps (Figure 1). The pericentriolar Golgi stacks are first fragmented into mitotic Golgi blobs, which undergo subsequent disassembly and appear as a haze. The conversion of the pericentriolar Golgi stacks into Golgi blobs has been reconstituted in permeabilized cells [20]. Two components have been identified that mediate this conversion: the mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) and polo-like kinase 1 (Plk1) [20,21]. The form of MEK1 involved in the Golgi fragmentation process has a conformation that is distinct from that used in the non-mitotic signalmediated pathway [14]. Specifically, it does not require its amino-terminal eight amino acids that are necessary for the binding and activation of the downstream target proteins extracellular-signal-regulated kinase (ERK) 1/ ERK2 (which are MAPKs) [14]. This is further supported by the recent findings that MEK1 undergoes a cdc2-kinase-dependent cleavage at its amino terminus, which prevents ERK activation and is required for progression into mitosis [22]. We have found that mitotically activated MEK1 is found on Golgi membranes in late prophase, and proposed that this association triggers Golgi fragmentation [23].

Figure 1

Pericentriolar Golgi apparatus

Golgi haze

Golgi blobs

Vesiculation cdc2

PIk1, MEK1 Nucleus

Redistribution into the ER Step 1

Step 2

Blocking this step inhibits

The significance of this

entry into mitosis

disassembly step is not known

Current Opinion in Cell Biology

The pericentriolar stacks of Golgi cisternae undergo two sequential fragmentation reactions during mitosis in mammalian cells. Plk1 and MEK1 are required for converting these stacks into tubulo-reticular elements (Golgi blobs). These Golgi blobs undergo further disassembly. The extent to which Golgi blobs are processed in this second disassembly reaction is controversial. The current extreme proposals are conversion into small vesicles by a process that requires cdc2 kinase, or fusion with the ER. It has been reported recently that preventing the fragmentation of the pericentriolar Golgi stacks into Golgi blobs inhibits entry of mammalian cells into mitosis. It would be most valuable to ascertain the effect of blocking the second disassembly step on the fate of the Golgi membranes in vivo and on the progression of cells through mitosis. www.current-opinion.com

Current Opinion in Cell Biology 2003, 15:462–467

464 Membranes and organelles

The Golgi-associated target(s) of MEK1 in vivo remain unclear. A monotyrosine-phosphorylated form of ERK induces Golgi fragmentation in intact cells [24]. However, it is not known whether the mitotic MEK1 can generate this specific form of ERK. An ERK or ERK-like protein has been found on the Golgi membranes, but its identity and significance in the Golgi fragmentation process is not known [20]. The Golgi-associated protein GRASP55 (Golgi reassembly stacking protein 55) is implicated in stacking Golgi cisternae and is phosphorylated by ERK2 [25,26]. It is conceivable that MEK1 (via the Golgiassociated ERK-like protein) phosphorylates GRASP55 in late prophase and unstacks Golgi cisternae. The mitotic kinase Plk1, which is important for the maturation of centrosomes, is also required for Golgi fragmentation. Its in vivo substrate on the Golgi membranes has not been established convincingly. An additional component of Golgi fragmentation in mitosis could be the recently identified Golgi-associated protein golgin-84 [27]. This protein is phosphorylated in vitro by mitotic cytosol and its depletion by RNA interference results in the separation of Golgi stacks from each other and their dispersal in the cytoplasm [28]. It is possible that golgin-84 is the substrate of a mitotic kinase and involved in the process by which Golgi stacks are severed from each other. Although several issues remain unresolved, we are confident that reactions mediated by these components separate Golgi cisternae from each other and from the pericentriolar connection in vivo. This generates tubuloreticular elements (mitotic Golgi blobs) that are dispersed throughout the cell. Incubation of rat liver Golgi membranes with mitotic cytosol generates smaller Golgi fragments (step two of the overall fragmentation process [Figure 1]) [29]. Cdc2 kinase is required for this reaction, and Shorter and Warren [4] proposed an elegant scheme for the conversion of Golgi stacks into small vesicles. The cis-Golgi protein GM130 plays a key role in this scheme. In vitro studies have revealed that GM130 binds to the COP1-vesicleassociated protein p115 [30]. This interaction is proposed to tether COP1 vesicles to Golgi cisterna [4]. GM130 is phosphorylated by cdc2 kinase, which abrogates GM130 binding to p115 in vitro [31]. These in vitro findings lend support to the proposal that Golgi membranes are converted into small vesicles via a block in the GM130–p115 interaction. Recent in vivo findings, however, challenge the validity of this scheme. For example, disrupting the interaction between GM130 and p115 in vivo does not affect Golgi organization [32]. Moreover, depletion of GM130 through use of reagents that cause its intracellular degradation neither affect Golgi organization nor the entry and exit of cells from mitosis [28]. These results suggest that the vesiculation of the Golgi apparatus is not solely caused by cdc2-kinase-dependent phosphorylation of GM130. Other processes must also participate. Current Opinion in Cell Biology 2003, 15:462–467

Lippincott-Schwartz and colleagues [9] argue that Golgi membranes are not in the form of vesicles, but fused with the ER owing to an imbalance in protein transport between the ER and the Golgi. It is known that protein transport out of the ER is blocked in mitosis [12,13,33]. Unfortunately, nothing is known about the kinetics of retrograde transport during mitosis, which makes it difficult to assess the validity of this model. It is important to note that endocytosis is blocked during mitosis, but there is no appreciable change in the organization of endosomes or lysosomes [34]. Thus, compartments that communicate via bidirectional transport reactions do not always lose their organization solely because one arm does not participate. We offer an alternative view that may explain the apparent disagreements in the field. We propose that mitotic Golgi blobs are fully competent for retrograde transport. A (small?) fraction of Golgi proteins are transported to the ER and kept there because of a block in ER export. Cells progress through mitosis and those that spend more than the required amount of time in between pro-metaphase and anaphase display higher levels of Golgi proteins in the ER. Another important factor that could influence the levels of Golgi proteins in the ER is the timing of the ER exit block. These two properties could explain the cell-type-specific differences in the reported levels of mitotic haze (Ptk1 cells compared with HeLa cells, see above). This could also explain localization of 20% of Golgi enzymes to the ER exit sites, as observed by Lucocq and colleagues [13], our findings of Golgi in the form of tubuloreticular elements [14] and the clusters (and a small amount of haze) observed by Warren and colleagues [10]. The mitotic blobs are the major partitioning units of the Golgi membranes. When cells exit mitosis, Golgi membranes in the form of mitotic Golgi blobs (separated from the ER) undergo extensive remodeling to re-form stacks. Polymerization of cytoplasmic microtubules drives the relocation of the stacks to the pericentriolar region; the Golgi proteins that were in the ER are brought to the newly formed Golgi apparatus by protein transport. This proposal is worth considering, given the level of discordance regarding the state of the ER–Golgi interface during and after mitosis.

Reason(s) for Golgi fragmentation A simple scheme that was presented over two decades ago is that Golgi fragmentation generates numerous small units, which are partitioned into the daughter cells. In post-mitotic cells, these units reassemble into a Golgi apparatus that is functionally and structurally equivalent to the maternal Golgi apparatus [35]. In this model, the process of fragmentation is seen as necessary for the partitioning of the maternal Golgi apparatus. Although this remains a valid proposal, a formal proof has been lacking. What would happen to a cell in which fragmentation of the pericentriolar Golgi apparatus is inhibited? www.current-opinion.com

Cell-cycle-specific Golgi fragmentation Colanzi et al. 465

There are two potential outcomes: the cell divides and one of the daughter cells acquires the maternal Golgi apparatus; the other cell dies, especially if there is no potential of de novo Golgi biogenesis. The second possibility is that the cell does not enter or exit mitosis. The ultimate fate under this condition would be cell death. It was recently reported that interfering with the fragmentation of pericentriolar Golgi membranes prevents entry into mitosis [36]. Antibodies to the Golgi-associated protein GRASP65 or a fragment of GRASP65 that contains the antibody-binding site inhibited Golgi fragmentation in a semi-intact cell assay. Injection of these reagents into NRK cells prevented entry into mitosis and the cells were arrested in G2. Interestingly, fragmentation of the Golgi membranes by the use of nocodazole (which depolymerises microtubules and causes dispersion of Golgi stacks throughout the cytoplasm) or brefeldin A (which causes Golgi membranes to fuse with the ER) in cells that had been injected with the GRASP65-specific reagents alleviated the block and the cells entered mitosis. These results tell an interesting tale; namely, that fragmentation of the Golgi is necessary for entry into mitosis. Moreover, the position of the pericentriolar Golgi apparatus appears to be an important sensor for regulating progression of cells into mitosis. This proposal is highly provocative, as it alludes to the existence of a mechanism by which cells regulate entry into mitosis, independent and in addition to the controls (checkpoints) that are activated through DNA damage [37]. This brings us back to the important issue of why the mammalian cells have evolved a mechanism to concentrate Golgi membranes in the pericentriolar region. Other cell types do a perfect job without concentrating these membranes in one specific cellular site. This specific locale is not important for protein sorting and transport, the known Golgi-associated function. Mammalian cells must concentrate Golgi membranes in the pericentriolar region for additional functions. One such function is to use these membranes as a sensor to ensure the proper divisibility of the cytoplasmic machineries in preparation for entry into mitosis. It makes no sense to have the nuclear material transferred to daughter cells without the compartment designated for sorting and transport of proteins for nuclear-encoded gene products. Thus, just as DNA damage triggers cells to abort entry into mitosis, a defect in Golgi fragmentation might elicit a similar response. This proposal is appealing at present, but will require further substantiation. Why do mitotic Golgi blobs undergo further processing into vesicles or redistribution into the ER during mitosis? As discussed earlier, the partitioning units of Golgi membranes in plants, Drosophila and parasites appear to be either intact stacks or smaller stacks. So why are mitotic Golgi blobs not sufficient as partitioning units in the www.current-opinion.com

mammalian cells? Is further disassembly necessary? Would blocking the conversion of blobs into haze influence Golgi partitioning or entry or exit from mitosis? It remains a possibility that the complete conversion to vesicles or fusion with the ER (whichever turns out to be true), which is a kinetic process, is dictated by the time a given cell spends between prometaphase and early anaphase, as described above. The Golgi fragmentation is, after all, an enzymatic reaction, and consequently any event that causes a block or delay in mitosis will exaggerate the disassembly process. We need to address this issue to better understand the significance of the second fragmentation step.

Golgi membranes: an emerging platform for a variety of cellular reactions Golgi membranes play an accepted role in the intracellular sorting and transport of proteins. They are also host to several proteins that are involved in signaling and apoptosis [38]. It is conceivable that these proteins reside on Golgi membranes in a dormant form, until the receipt of a signal. The signal would then activate the Golgi residents, which would either dissociate and be delivered to their respective site of action or act within the confines of the Golgi membrane. For example, a small fraction of the GTPase Ras is Golgi-associated [39]. Upon activation, Ras recruits the downstream effector Raf-1 to the Golgi membranes to form a highly specific signaling environment. Other signaling events on Golgi membranes require additional scaffolding proteins, for example AKAP450, which recruits several other signaling molecules [40]. An additional example of a protein that requires the Golgi apparatus for activation is the fused protein product FIG-ROS, a potent oncogene product that acquires oncogenic transformation capacity once it is targeted to the Golgi apparatus [41]. The Golgi-associated protein golgin-160 is cleaved by a resident caspase and this is somehow involved in triggering apoptosis [42]. It is thus reasonably safe to bet that mammalian cells have evolved to exploit Golgi membranes and their unique cellular locale to regulate several key cellular processes: entry into mitosis and apoptosis currently being two obvious ones. The list, however, is likely to grow, and it would be best to keep an open mind regarding the dynamics of the Golgi membranes and their involvement in cellular processes far beyond the sorting and transport of proteins.

Conclusions The control of progression into mitosis via the pericentriolar position of the Golgi apparatus is a novel concept. Protein kinases MEK1 and Plk are essential for fragmentation of the pericentriolar Golgi apparatus. An understanding of upstream activators and downstream targets of these kinases should help unravel the mechanism through which the locale of the Golgi apparatus is exploited by cells to enter mitosis. Current Opinion in Cell Biology 2003, 15:462–467

466 Membranes and organelles

Acknowledgements We thank Alberto Diaz-Anel for help with the figure and Matthew Kinseth for comments on the manuscript. Work in the Malhotra laboratory is supported by grants from National Institutes of Health and Human Frontiers Science Program. The first two authors contributed equally to this review.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Preuss D, Mulholland J, Franzusoff A, Segev N, Botstein D: Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol Biol Cell 1992, 3:789-803.

13. Prescott AR, Farmaki T, Thomson C, James J, Paccaud JP, Tang BL, Hong W, Quinn M, Ponnambalam S, Lucocq J: Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates. Traffic 2001, 2:321-335. 14. Colanzi A, Deerinck TJ, Ellisman MH, Malhotra V: A specific  activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis. J Cell Biol 2000, 149:331-339. The authors report that the conformation of mitotically activated MEK1 is different from the interphase MEK1. Additionally, the mitotically activated form of MEK1 fragments Golgi membranes even when its ability to activate cytosolic ERKs is inhibited. This provides further support for the proposal that MEK1 is specifically activated during mitosis and interacts with a novel Golgi-associated target. 15. Nelson WJ: W(h)ither the Golgi during mitosis? J Cell Biol 2000, 149:243-248.

2.

Nebenfuhr A, Staehelin LA: Mobile factories: Golgi dynamics in plant cells. Trends Plant Sci 2001, 6:160-167.

16. Jokitalo E, Cabrera-Poch N, Warren G, Shima DT: Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum. J Cell Biol 2001, 154:317-330.

3.

Stanley H, Botas J, Malhotra V: The mechanism of Golgi segregation during mitosis is cell-type-specific. Proc Natl Acad Sci USA 1997, 94:14467-14470.

17. Seemann J, Pypaert M, Taguchi T, Malsam J, Warren G: Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells. Science 2002, 295:848-851.

4.

Shorter J, Warren G: Golgi architecture and inheritance. Annu Rev Cell Dev Biol 2002, 18:379-420.

5.

Benchimol M, Ribeiro KC, Mariante RM, Alderete JF: Structure and division of the Golgi complex in Trichomonas vaginalis and Tritrichomonas foetus. Eur J Cell Biol 2001, 80:593-607.

18. Takizawa PA, Yucel JK, Veit B, Faulkner DJ, Deerinck T, Soto G, Ellisman M, Malhotra V: Complete vesiculation of Golgi membranes and inhibition of protein transport by a novel sea sponge metabolite, ilimaquinone. Cell 1993, 73:1079-1090.

6.

Pelletier L, Stern CA, Pypaert M, Sheff D, Ngo HM, Roper N, He CY, Hu K, Toomre D, Coppens I et al.: Golgi biogenesis in Toxoplasma gondii. Nature 2002, 418:548-552.

7.

Burke B, Griffiths G, Reggio H, Louvard D, Warren G: A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J 1982, 1:1621-1628.

8. 

Thyberg J, Moskalewski S: Reorganization of the Golgi complex in association with mitosis: redistribution of mannosidase II to the endoplasmic reticulum and effects of brefeldin A. J Submicrosc Cytol Pathol 1992, 24:495-508. Immunoelectron microscopy revealed that Golgi enzymes are redistributed into the ER in mitotic cells. This was the beginning of the controversy regarding the fate of the Golgi membranes during mitosis in mammalian cells. 9. 

Zaal KJ, Smith CL, Polishchuk RS, Altan N, Cole NB, Ellenberg J, Hirschberg K, Presley JF, Roberts TH, Siggia E et al.: Golgi membranes are absorbed into and re-emerge from the ER during mitosis. Cell 1999, 99:589-601. Live-imaging, photo bleaching revealed the presence of Golgi enzymes in the ER in mitotic cells. The photo-bleaching experiment is the strongest in this manuscript. It would be valuable to test the kinetics of fluorescence recovery of mitotic cells with cells containing vesiculated Golgi membranes. For example, treatment of cells with the drug ilimaquinone converts Golgi membranes into small vesicles. If Golgi membranes are fused with the ER, as argued by the authors, then there should be a difference in fluorescence recovery between IQ treated cells and mitotic cells. 10. Shima DT, Haldar K, Pepperkok R, Watson R, Warren G: Partitioning of the Golgi apparatus during mitosis in living HeLa cells. J Cell Biol 1997, 137:1211-1228. 11. Jesch SA, Mehta AJ, Velliste M, Murphy RF, Linstedt AD: Mitotic  Golgi is in a dynamic equilibrium between clustered and free vesicles independent of the ER. Traffic 2001, 2:873-884. Mitotic cells were fractionated by ultracentrifugation. ER- and Golgispecific markers were found in different fractions, suggesting that these two compartments remain separate during mitosis. It cannot be ruled out, however, that the cells used in these experiments were only those that contain Golgi membranes diffusely dispersed (prometaphase to early anaphase). Golgi membranes in the preceding mitotic stages (late prophase to early metaphase) are known to be separate from the ER and would definitely show Golgi and ER membranes in different fractions by the procedure used by the authors. 12. Hammond AT, Glick BS: Dynamics of transitional endoplasmic reticulum sites in vertebrate cells. Mol Biol Cell 2000, 11:3013-3030. Current Opinion in Cell Biology 2003, 15:462–467

19. Mironov AA, Polishchuk RS, Luini A: Visualizing membrane traffic in vivo by combined video fluorescence and 3D electron microscopy. Trends Cell Biol 2000, 10:349-353. 20. Acharya U, Mallabiabarrena A, Acharya JK, Malhotra V:  Signaling via mitogen-activated protein kinase kinase (MEK1) is required for Golgi fragmentation during mitosis. Cell 1998, 92:183-192. Mitotic Golgi fragmentation was reconstituted in permeabilized NRK cells. This semi-intact cell assay led to the identification of MEK1 kinase, a key component of the mitogen-activated protein kinase, signaling cascade, in the Golgi fragmentation process. Interestingly, cytosolic ERK1 and ERK2, the known MEK1 substrates, were not required for this reaction. A Golgi-bound ERK or an ERK-like protein was revealed by fluorescence microscopy. 21. Sutterlin C, Lin CY, Feng Y, Ferris DK, Erikson RL, Malhotra V:  Polo-like kinase is required for the fragmentation of pericentriolar Golgi stacks during mitosis. Proc Natl Acad Sci USA 2001, 98:9128-9132. Using the semi-intact cell assay described by Acharya et al. (1998)  [20 ], polo-kinase 1, a mitotic kinase with an important role in the maturation of centrosomes, was shown to be required for mitotic Golgi fragmentation. 22. Harding A, Giles N, Burgess A, Hancock JF, Gabrielli B: Mechanism of mitosis-specific activation of MEK1. J Biol Chem 2003, DOI 301015200. 23. Colanzi A, Suetterlin C, Malhotra V: RAF-1 activated MEK1 is  found on the Golgi apparatus in late prophase and is required for Golgi fragmentation during mitosis. J Cell Biol 2003, 161:27-32. The authors report that mitotically activated MEK1 is localized to the Golgi membranes during late prophase. This localization coincides with the initiation of the Golgi fragmentation process. However, the mechanism by which mitotic MEK1 is bound to the Golgi in late prophase and its Golgiassociated targets are not known. 24. Cha H, Shapiro P: Tyrosine-phosphorylated extracellular signal–regulated kinase associates with the Golgi complex during G2/M phase of the cell cycle: evidence for regulation of Golgi structure. J Cell Biol 2001, 153:1355-1367. 25. Jesch SA, Lewis TS, Ahn NG, Linstedt AD: Mitotic phosphorylation of Golgi reassembly stacking protein 55 by mitogen-activated protein kinase ERK2. Mol Biol Cell 2001, 12:1811-1817. 26. Shorter J, Watson R, Giannakou ME, Clarke M, Warren G, Barr FA: GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system. EMBO J 1999, 18:4949-4960. www.current-opinion.com

Cell-cycle-specific Golgi fragmentation Colanzi et al. 467

27. Diao A, Rahman D, Pappin DJ, Lucocq J, Lowe M: The coiled coil membrane protein golgin-84 is a novel Rab effector required for Golgi ribbon formation. J Cell Biol 2003, 160:201-212. The Golgi-associated protein golgin-84 is shown to be required for linking stacks of Golgi cisternae in the pericentriolar region. Loss of golgin-84 resulted in disassembly of the Golgi apparatus into stacks, which were found dispersed throughout the cell. Golgin-84 is phosphorylated in mitotic cells; however, the significance of this phosphorylation in mitotic Golgi dynamics is not yet known.

34. Bergeland T, Widerberg J, Bakke O, Nordeng TW: Mitotic  partitioning of endosomes and lysosomes. Curr Biol 2001, 11:644-651. In this paper, the authors show that endosomes and lysosomes remain intact during mitosis in mammalian cells. It is known that endocytosis is blocked during mitosis. A block in arrival of proteins into the endosomal pathway does not perturb the organization of the endosomes during mitosis. We use this information to raise an issue that imbalance of protein transport from ER to Golgi might not be the (only) reason for extensive Golgi fragmentation in mitosis.

28. Puthenveedu MA, Linstedt AD: Evidence that Golgi structure  depends on a p115 activity that is independent of the vesicle tether components giantin and GM130. J Cell Biol 2001, 155:227-238. The authors report that removal of GM130 from cells does not affect the overall organization of the Golgi apparatus. This finding challenges the role of GM130 as a key regulator of the organization of the Golgi apparatus.

35. Warren G: Membrane partitioning during cell division. Annu Rev Biochem 1993, 62:323-348.

29. Misteli T, Warren G: COP-coated vesicles are involved in the  mitotic fragmentation of Golgi stacks in a cell-free system. J Cell Biol 1994, 125:269-282. The authors used a cell-free assay to show that fragmentation of the Golgi membranes by mitotic cytosol is through a COP1-dependent process. However, it is not known whether inhibiting COP1 activity in intact cells affects Golgi fragmentation during mitosis. 30. Nakamura N, Lowe M, Levine TP, Rabouille C, Warren G: The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Cell 1997, 89:445-455. 31. Lowe M, Rabouille C, Nakamura N, Watson R, Jackman M,  Jamsa E, Rahman D, Pappin DJ, Warren G: Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis. Cell 1998, 94:783-793. An in vitro assay that reconstitutes fragmentation of the isolated Golgi membrane into smaller elements upon incubation with mitotic cytosol was found to be cdc2 kinase-dependent. The Golgi associated protein GM130 is phosphorylated by cdc2 kinase. However, the significance of cdc2 kinase dependent phosphorylation of GM130 in mitosis specific Golgi dynamics is not known. 32. Seemann J, Jokitalo EJ, Warren G: The role of the tethering proteins p115 and GM130 in transport through the Golgi apparatus in vivo. Mol Biol Cell 2000, 11:635-645. 33. Featherstone C, Griffiths G, Warren G: Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells. J Cell Biol 1985, 101:2036-2046.

www.current-opinion.com

36. Sutterlin C, Hsu P, Mallabiabarrena A, Malhotra V: Fragmentation  and dispersal of the pericentriolar Golgi complex is required for entry into mitosis in mammalian cells. Cell 2002, 109:359-369. The authors report that inhibiting fragmentation of pericentriolar Golgi apparatus prevents entry into mitosis. It is suggested that position of Golgi membranes acts as a sensor for regulating entry into mitosis. When fragmentation is inhibited, cells activate a machinery that inhibits progression into mitosis. 37. Melo J, Toczyski D: A unified view of the DNA-damage checkpoint. Curr Opin Cell Biol 2002, 14:237-245. 38. Donaldson JG, Lippincott-Schwartz J: Sorting and signaling at the Golgi complex. Cell 2000, 101:693-696. 39. Chiu VK, Bivona T, Hach A, Sajous JB, Silletti J, Wiener H, Johnson RL II, Cox AD, Philips MR: Ras signalling on the endoplasmic reticulum and the Golgi. Nat Cell Biol 2002, 4:343-350. 40. Sillibourne JE, Milne DM, Takahashi M, Ono Y, Meek DW: Centrosomal anchoring of the protein kinase CK1delta mediated by attachment to the large, coiled-coil scaffolding protein CG-NAP/AKAP450. J Mol Biol 2002, 322:785-797. 41. Charest A, Kheifets V, Park J, Lane K, McMahon K, Nutt CL, Housman D: Oncogenic targeting of an activated tyrosine kinase to the Golgi apparatus in a glioblastoma. Proc Natl Acad Sci USA 2003, 100:916-921. 42. Mancini M, Machamer CE, Roy S, Nicholson DW, Thornberry NA,  Casciola-Rosen LA, Rosen A: Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis. J Cell Biol 2000, 149:603-612. The authors report that caspase-2 cleaves the Golgi-associated protein golgin-160. Expression of a golgin-160 mutant lacking the unique caspase-2 cleavage site delayed Golgi disassembly by the apoptosis inducing agent staurosporine. This manuscript suggests that Golgi-associated proteins are able to participate (or even induce) apoptosis.

Current Opinion in Cell Biology 2003, 15:462–467