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Vol. 17, Issue 12, 5372-5380, December 2006
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Department of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093
Submitted July 3, 2006;
Revised August 18, 2006;
Accepted October 5, 2006
Monitoring Editor: Francis Barr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Integral membrane proteins and proteins that are secreted from the cell are translated into the endoplasmic reticulum (ER), where they are properly folded, and transported to the Golgi apparatus. Within Golgi membranes proteins are sequentially modified by the polarized array of Golgi enzymes and then transported to their respective destinations. Brefeldin A (BFA), a fungal metabolite that blocks ER-Golgi transport (Fujiwara et al., 1988), induces the relocation of Golgi enzymes into the ER (Lippincott-Schwartz et al., 1989). Preventing ER exit through the overexpression of a dominant-negative form of sar1, a GTPase that recruits the COPII coat to ER exit sites (Barlowe et al., 1994), also relocates Golgi enzymes into the ER (Storrie et al., 1998; Zaal et al., 1999; Miles et al., 2001; Ward et al., 2001). These studies demonstrate that Golgi enzymes have the capacity to quickly redistribute into the ER under artificial conditions, but they may not depict what happens normally, when the organization of the secretory pathway is not perturbed. Nonetheless, based on these findings, it was proposed that Golgi enzymes constitutively cycle through the ER (Zaal et al., 1999; Miles et al., 2001; Ward et al., 2001). Further investigation through use of dominant-negative sar1 reagents has revealed that ER recycling is a general property of all Golgi proteins (Miles et al., 2001). Implicit to this idea is the dependence of Golgi function on the perpetual retrieval of Golgi components from the ER, which opposes the status of the Golgi as an independent organelle.
The dynamics of the Golgi apparatus are determined by the collective behavior of its resident enzymes. Thus, the relationship between the Golgi and the ER can be ascertained by determining the rate at which Golgi enzymes associate with the ER. Previously, researchers have used fluorescence recovery after photobleaching techniques to achieve this (Zaal et al., 1999; Miles et al., 2001; Ward et al., 2001). However, in these studies conclusions were based on the analysis of very few cells (n = 3 cells; Miles et al., 2001). Moreover, in some studies a clearly detectable ER-specific pool of a fluorescently tagged Golgi reporter was required in order to measure ER recycling with this method (Zaal et al., 1999; Miles et al., 2001). In our experiments Golgi enzymes are only detected in the ER if highly overexpressed. Because the behavior of highly overexpressed Golgi enzymes may not accurately emulate that of endogenous enzymes, we made a point to execute the experiments presented in this report under more physiological expression levels. We have investigated the ER recycling of resident Golgi proteins through use of a procedure that captures Golgi enzymes in the ER (Pecot and Malhotra, 2004). This method exploits the conditional interaction of two proteins and thus allows the dynamics of Golgi enzymes to be observed without photobleaching cells or disrupting the secretory pathway. The FK506-binding protein (FKBP) and the FKBP-rapamycin–associated protein (FRAP) only interact in the presence of rapamycin, a small molecule. Rapamycin specifically binds to FKBP (Wiederrecht et al., 1991) and FRAP binds to the FKBP-rapamycin complex (Brown et al., 1994; Sabatini et al., 1994). In our procedure, FKBP is fused to a Golgi enzyme, and FRAP is attached to an ER-retained protein. If the FKBP-tagged Golgi enzyme ever visits the ER it can be trapped there in the presence of rapamycin by the ER-FRAP chimera (Figure 1A). We have shown previously that a Golgi enzyme (sialyl-transferase) fused to FKBP can be trapped quickly and efficiently in the ER of BFA-treated cells via this method (Pecot and Malhotra, 2004). Through use of this procedure we demonstrated that Golgi membranes remain segregated from the ER during mitosis in mammalian cells (Pecot and Malhotra, 2004). Here we use the ER-trapping method to investigate the relationship between the Golgi apparatus and the ER in nondividing cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning and Constructs
ST-FKBP and Ii-FRAP were previously described (Pecot and Malhotra, 2004). GFP-ERGIC-53 was a gift from Dr. Hauri (Ben-Tekaya et al., 2005). One tandem repeat of full-length FKBP was cloned into the BglII restriction site of GFP-ERGIC-53, generating GFP-FKBPx2-ERGIC-53 (FKBP-E53). M2-FKBP was constructed by cloning the first 100 amino acids of mouse alpha-mannosidase II (base pairs 88-307) into the HindIII and EcoRI restriction sites of pEGFP-N3. The entire FKBP coding region was then cloned into the EcoRI and BamHI restriction sites of this vector, leaving a C-terminal GFP tag.
Cell Culture. HeLa cells (ATCC, Manassas, VA) were grown at 37°C with 7% CO2 in DMEM supplemented with 10% FBS, and 2 mM L-glutamine.
De-convolution Microscopy. Images were captured with a Nikon Eclipse TE 2000-U inverted microscope (Melville, NY) and a Photometrics CoolSNAP HQ camera (A. G. Heinz, Tucson, AZ). Image acquisition was run by Metamorph software (Universal Imaging, West Chester, PA), and images were de-convolved by AutoDeblur and AutoVisualize 9.3 software (AutoQuant Imaging, Watervliet, NY). In general, using a 60x objective lens (NA 1.4), 21 optical sections per cell spaced by 0.1 µm were taken. The best optical section for each set of data were used for publication. Exposure times were set such that the camera response was in the linear range for each fluorophore.
Transfections. HeLa cells were split into 6-cm plates the night before (600–900K cells/plate). In the morning the cells were between 80 and 95% confluent. Transfections were performed in the morning via Lipofectamine, 2000 Reagent (Invitrogen, Carlsbad, CA). Twenty microliters of Reagent plus 10 µg of total DNA (FKBP-E53, M2-FKBP, or ST-FKBP + Ii-FRAP) was added to each 6-cm plate, and transfections were allowed to proceed for at least 6 h. In the evening the cells were split onto coverslips in 6-cm plates and allowed to attach for 12–18 h.
ER-Trapping Procedure.
After transfection, cells were treated with media only, media + cycloheximide (CHX, 100 µg/ml) + BFA (10 µg/ml), or media + CHX + BFA + rapamycin (Rap, 200 nM) for 10 min at 37°C. At this point coverslips were fixed to determine the percentage of cells that had completely redistributed M2-FKBP into the ER. Cotransfected cells expressing high amounts of Ii-FRAP were counted (
400 cells/experiment). The remaining coverslips were washed three times with PBS and incubated for 2 h at 37°C with media + CHX, or media + CHX + Rap. The coverslips were then fixed (4% formaldehyde in PBS) for 10 min at room temperature, followed by treatment with blocking buffer (2% FBS, 0.1% TX-100, and 0.05% NaN3 in PBS) for 30 min at room temperature. Ii-FRAP was labeled with a monoclonal HA antibody (1:1K in PBS, 1 h room temperature). All secondary antibodies (Molecular Probes) were used at a dilution of 1:2K in PBS (20 min room temperature). After antibody staining, cotransfected cells were visualized by fluorescence microscopy (GFP constructs were visualized via GFP fluorescence). ER trapping was determined in cells expressing large amounts of Ii-FRAP. Only the complete redistribution of M2-FKBP into the ER was counted as trapping. Approximately 300 cells were scored per experiment (3 independent experiments).
15°C Experiment. HeLa cells expressing Ii-FRAP and FKBP-E53 were incubated at 37 or 15°C for 3 h in sealed 24-well plates with culture medium containing 25 mM HEPES. The cells were then fixed and prepared for fluorescence microscopy as described above.
Recycling Experiments.
HeLa cells were transfected as described above. Transfected cells were treated with media + CHX (100 µg/ml), or media + CHX + Rap (200 nM) for various periods of time. At designated time points the cells were fixed and prepared for fluorescence microscopy (as described above). Cotransfected cells expressing high amounts of Ii-FRAP were scored for the recycling of Golgi chimeras to the ER. Only complete relocation into the ER was counted as recycling. Approximately 200 cells were analyzed per experiment with M2-FKBP (at least 3 independent experiments), 55 cells per experiment with FKBP-E53 (at least 2 independent experiments), and 80 cells per experiment with ST-FKBP (2 independent experiments).
Quantitation of Partial Trapping for Recycling Experiments. Recycling experiments were performed as described above. The percentage of cotransfected cells displaying complete Golgi localization (No Trapping), complete ER localization (Complete Trapping), weak partial ER localization (weak ER staining), or strong partial ER localization (strong ER staining) for M2-FKBP or ST-FKBP after 8 h or 16 h in the presence of CHX ± Rap was determined.
Nocodazole Experiments Cells expressing Ii-FRAP and M2-FKBP were treated with media + CHX (100 µg/ml), media + CHX + nocodazole (Noc, 1 ìM), or media + CHX + Noc + Rap (200 nM) at 37°C for 1.5–2 h. Cells were prepared for fluorescence microscopy as described above, except that Hoechst (1:25K) was added to the secondary antibody incubation. The effects of ER trapping on the appearance of Noc-induced Golgi fragments were observed in cotransfected cells expressing large amounts of Ii-FRAP. Cells were scored based on their having or not having small Golgi fragments. Approximately 100 cells were analyzed per experiment (4 independent experiments).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and E53 resides in the ERGIC (Schweizer et al., 1988). E53 rapidly cycles through the ER as it is a cargo receptor for the transport of glycoproteins between the ER and ERGIC (Hauri et al., 2000). For this reason E53 is an excellent molecule with which to compare the ER association of Golgi enzymes. When expressed in HeLa cells M2-FKBP and FKBP-E53 localize to the Golgi and ERGIC, respectively (Figure 1B). The GFP-E53 construct, from which FKBP-E53 is derived, was previously constructed and utilized to investigate the dynamics of the ERGIC in living cells and is thus a proven marker of the ERGIC (Ben-Tekaya et al., 2005). Ii-FRAP and ST-FKBP (Figure 1B) have been previously described (Pecot and Malhotra, 2004). Briefly, the FKBP-rapamycin binding domain of FRAP (Chen et al., 1995) was fused to an ER-retained version of the invariant chain protein (Iip33; Schutze et al., 1994; Ii-FRAP), and the Golgi localization domain of sialyl-transferase (a late Golgi enzyme) was fused to FKBP and GFP (ST-FKBP). Iip33 cannot be detected in post-ER compartments with biochemical methods (Schutze et al., 1994) or by fluorescence microscopy (Schutze et al., 1994; Figure 1B), indicating that it does not leave the ER. In addition, unlike Sec31 Ii-FRAP does not accumulate in proliferating ERGIC elements at 15°C (Figure 2). At this temperature ERGIC elements undergo proliferation (Hauri and Schweizer, 1992; Saraste and Kuismanen, 1992) and accumulate molecules that cycle between the ER and Golgi (Schweizer et al., 1990; Jackson et al., 1993; Tang et al., 1993; Nickel et al., 1997; Hay et al., 1998) including components of ER exit sites (Hammond and Glick, 2000).
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), but does not hinder the efficiency of ER trapping in our experiments. At designated time points the cells were fixed and the localization of FKBP-E53 was observed by fluorescence microscopy. We discovered that the recycling of FKBP-E53 to the ER takes place in three stages (Figure 3A). Stage I is depicted by the loss of a compact peri-nuclear structure and the formation of an ER-like reticular pattern. More than 90% of observed cells display this phenotype after 5 min (Figure 3B). In stage II, the entire visible pool of FKBP-E53 has been converted into a reticular ER pattern. At this point the cycling of FKBP-E53 into the ER is complete as there is no longer evidence of any other structure by de-convolution microscopy. After 15 min, more than 60% of the observed cells exhibit this staining for FKBP-E53 (Figure 3B). Interestingly, although at this time FKBP-E53 is completely in the ER, it has not yet spread throughout the entire ER network, as observed by Ii-FRAP staining. Stage III signifies the complete dispersal of FKBP-E53 throughout the ER network. In 30 min approximately half of the observed cells are stage III, and by 1 h nearly all the cells display this phenotype (Figure 3B). These results confirm that FKBP-E53 rapidly cycles through the ER and that the ER-trapping procedure is capable of capturing such proteins.
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80% had trapped the entire pool of M2-FKBP in the ER after BFA release in the presence of rapamycin (Figure 4B). These results, combined with the data obtained for FKBP-E53 demonstrate that the ER-trapping procedure is efficient and capable of capturing rapidly recycling proteins, thereby justifying its use as a method to determine if Golgi enzymes constitutively cycle through the ER.
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; Miles et al., 2001). However, after 16 h in the presence of rapamycin, 90% of the observed cells still displayed a clearly visible Golgi-specific pool of M2-FKBP (Figure 4A, Table 1). Similar results were recorded for cells coexpressing Ii-FRAP and ST-FKBP (Table 1). When M2-GFP (lacking FKBP coding region) was substituted for M2-FKBP in recycling experiments, no background recycling was observed (data not shown). Time points longer than 16 h could not be taken because long-term cycloheximide treatment induces apoptosis.
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Nocodazole-induced Golgi Dispersion Does Not Result from the Recycling of Golgi Membranes through the ER
In cells treated with Noc, a microtubule de-polymerizing agent, the Golgi apparatus becomes fragmented into individual stacks of cisternae that are dispersed throughout the cell (Thyberg and Moskalewski, 1985; Cole et al., 1996). It is thought that this fragmentation occurs via the recycling of Golgi membranes through the ER and their reemergence at areas adjacent to ER exit sites (Cole et al., 1996; Storrie et al., 1998; Figure 6A). We have tested this idea through use of the ER-trapping procedure. If it is true that Noc induces the fragmentation of Golgi membranes through ER recycling, then trapping Golgi enzymes in the ER should prevent the appearance of the scattered Golgi fragments (Figure 6A). If the ER-trapping procedure cannot prevent the formation of small Golgi elements then Noc-induced Golgi dispersion must transpire through an alternative mechanism. HeLa cells expressing M2-FKBP and Ii-FRAP were treated with Noc (1 µg/ml), and at assorted time points the cells were fixed and analyzed by fluorescence microscopy. After 1.5 h the typical Golgi-pattern represented by M2-FKBP began to breakdown into small, scattered elements that localized near ER exit sites (Figure 6B). When rapamycin was added with Noc and cycloheximide for 1.5–2 h, the appearance of small scattered fragments was prevented in 14% of cotransfected cells (Figure 6C). In the vast majority of cells (86%) small Golgi elements were clearly visible, indicating their appearance was not due to ER recycling (Figure 6, B and C). Our results suggest that the initial fragmentation and dispersal of the Golgi apparatus in response to Noc treatment primarily occurs through a mechanism that does not involve the recycling of Golgi membranes through the ER.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Miles et al., 2001; Ward et al., 2001). Exposing cells to BFA or dominant-negative forms of sar1 inhibits protein traffic out of the ER (Fujiwara et al., 1988; Storrie et al., 1998) and induces the accumulation of Golgi enzymes in the ER (Lippincott-Schwartz et al., 1989; Storrie et al., 1998; Miles et al., 2001; Ward et al., 2001), suggesting the existence of a Golgi-ER recycling pathway. However, both reagents may act nonspecifically due to the large amounts that must be added to cells to observe the desired effects. In support of this, BFA-mediated Golgi recycling requires microtubules (Lippincott-Schwartz et al., 1990), while sar1 induced recycling is microtubule independent (Storrie et al., 1998).
Fluorescence recovery after photobleaching techniques have been applied to demonstrate the rapid ER cycling of Golgi enzymes under physiological conditions (Zaal et al., 1999; Miles et al., 2001; Ward et al., 2001). However, as was discussed earlier few cells can be examined with this method, and in some reports the cells that are examined must overexpress the observed Golgi reporter in the ER (Zaal et al., 1999; Miles et al., 2001). We feel that these limitations make it difficult to make conclusions about the behavior of endogenous Golgi enzymes. Researchers have also utilized the temperature-sensitive features of the vesicular stomatitis virus G-protein (VSVG) to show that Golgi proteins recycle to the ER under normal conditions (Cole et al., 1998). The thermosensitive form of VSVG (VSVGtsO45) contains a temperature-sensitive mutation that causes it to aggregate and be retained in the ER at temperatures above 39.8°C (Gallione and Rose, 1985). Golgi proteins that have been fused with VSVGtsO45 have been shown to accumulate in the ER under these conditions (Cole et al., 1998), providing evidence for their constitutive recycling to the ER. However, it has been reported that at temperatures sufficient to arrest VSVGtsO45 in the ER, the budding of COPII-coated vesicles from the ER is prevented (Aridor et al., 1999) and general protein transport is inhibited (Trucco et al., 2004), demonstrating that these conditions do not represent a physiological situation. Furthermore, this study excluded Golgi enzymes and focused instead on Golgi-associated proteins known to behave dynamically as a result of their function in membrane trafficking (KDEL Receptor, TGN-38).
Because of the difficulty in accurately studying the behavior of Golgi enzymes, the relationship between the Golgi and the ER has been a contentious topic. For this reason, we developed the ER-trapping procedure (Figure 1A), which represents a simple, straightforward method to observe the association of Golgi enzymes with the ER under physiological circumstances. Previously we established that a late Golgi reporter (ST-FKBP) can be efficiently trapped in the ER when it visits there (Pecot and Malhotra, 2004). Here we have done the same for an early Golgi marker (M2-FKBP; Figure 4, A and B). We then determined if these reporters relocate to the ER over time. We discovered that although the entire pool of an ERGIC-53 reporter (FKBP-E53) is rapidly captured in the ER (30 min, Figure 3, A and B), our early and late Golgi markers remain stably associated with Golgi membranes (Figure 5, Table 1). Partial relocation of these proteins into the ER occurred in only a small percentage of cells (Figure 5B, Table 1), indicating that they do not constitutively cycle through the ER.
In this study we have also investigated the mechanism underlying Noc-induced Golgi fragmentation. Prior reports have claimed that Golgi fragmentation under these conditions occurs via the constitutive recycling of Golgi membranes through the ER (Cole et al., 1996; Storrie et al., 1998; Figure 6A). Based on these findings, it was also suggested that ER recycling is responsible for Golgi inheritance during cell division (Zaal et al., 1999; Altan-Bonnet et al., 2006), when Golgi membranes breakdown during spindle formation and reassemble in daughter cells (Warren, 1993). We have demonstrated that the appearance of Noc-dependent Golgi fragments cannot be prevented by trapping Golgi proteins in the ER (Figure 6, B and C), suggesting that fragmentation occurs through an alternative mechanism. These results support our prior finding that Golgi membranes remain segregated from the ER during mitosis in mammalian cells (Pecot and Malhotra, 2004).
We have demonstrated that both early and late Golgi enzymes remain stably associated with Golgi membranes and do not constitutively cycle through the ER. On the basis of this finding, we propose that the Golgi apparatus is a stable compartment that does not rely on the ER for its constant biogenesis. The results presented in this report combined with work regarding the fate of Golgi membranes during cell division (Jesch and Linstedt, 1998; Rossanese and Glick, 2001; Axelsson and Warren, 2004; Pecot and Malhotra, 2004) indicate that the Golgi apparatus remains independent from the ER throughout the life of the cell.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Vivek Malhotra (malhotra{at}biomail.ucsd.edu)
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-mannosidase II was fused to the FKBP coding region and GFP. ST-FKBP consists of the Golgi localization domain of sialyl-transferase appended to the FKBP coding region and GFP. Ii-FRAP is an ER-retained version of the invariant chain protein fused to the FKBP-rapamycin binding domain of FRAP and the HA epitope. All constructs were designed so that FKBP and FRAP domains are in the lumen of the Golgi or ER. In HeLa cells expressing Ii-FRAP FKBP-E53, M2-FKBP, and ST-FKBP (green, GFP) localize to the ERGIC, early Golgi, and late Golgi, respectively, and remain separate from ER-specific Ii-FRAP (red, HA).
