Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
* Author for correspondence (e-mail: wjb5{at}cornell.edu)
Accepted 12 April 2005
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Summary |
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Key words: Golgi complex, Endosome, Membrane tubules, Lysophospholipid acyltransferase, CI-976
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Introduction |
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Hydrolysis of PLs by a cytosolic PLA2 may cause a localized accumulation of lysophospholipids (LPLs) in the outer leaflet of the Golgi membrane bilayer (Brown et al., 2003). Such elevated LPL levels may increase membrane curvature leading to tubule formation (Christiansson et al., 1985
; Fujii and Tamura, 1979
). Therefore, Golgi tubulation might be mediated by controlling the lipid content of Golgi membranes. Lysophospholipid acyltransferases (LPATs) catalyze the reverse reaction of PLA2 by reacylating LPLs at the sn-2 position. In fact, an LPAT activity is known to be associated with the Golgi complex (Chambers and Brown, 2004
; Lawrence et al., 1994
), which could be involved in regulating the lipid composition and, thus, membrane structure, i.e., tubule formation. Interestingly, LPL acylation has been implicated as a common ingredient in several membrane fission events. The lysophosphatidic acid (LPA)-specific acyltransferase (LPAAT) activity of endophilin I has been reported to play a role in the formation of synaptic-like microvesicles (Schmidt et al., 1999
), although subsequent studies question whether the catalytic activity is required (Farsad et al., 2001
). An unrelated protein, C-terminal binding protein/BFA-ADP-ribosylated substrate (CtBP/BARS) was also found to exhibit LPAAT activity, and this enzyme is able to induce the fission of Golgi membrane tubules into vesicles during mitotic disassembly of the Golgi complex (Carcedo et al., 2004
; Weigert et al., 1999
). Again, however, the LPAAT activity of CtBP/BARS may facilitate but not be required for the fission reaction (Carcedo et al., 2004
). So, although no definitive examples of LPAATs that are required for fission have been uncovered, mounting evidence suggests that alterations in membrane bilayer lipid composition could have a direct effect on membrane structure and trafficking events (Brown et al., 2003
; Burger, 2000
; Corda et al., 2002
; Scales and Scheller, 1999
).
Because our previous studies implicated a role for PLA2-generated LPL production in Golgi membrane tubulation, we were interested to see what effect, if any, inhibition of LPL metabolism would have on the Golgi complex. These studies led to the identification of a novel inhibitory activity for a previously characterized antagonist of acyl-CoA cholesterol acyltransferase (ACAT), CI-976 (Harte et al., 1995). We found that CI-976 was a potent inhibitor of a Golgi-associated LPAT activity that displayed a preference for lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) (Chambers and Brown, 2004
; Drecktrah et al., 2003
). Remarkably, CI-976 caused a dramatic stimulation of Golgi tubule formation and redistribution of resident enzymes back to the ER. Importantly, pretreatment of cells with PLA2 antagonists inhibited CI-976 from inducing tubules, a result which strongly suggests that the CI-976 effect is dependent on the accumulation of LPLs, i.e. the substrate for LPATs. These results are consistent with the idea that lipid modifying enzymes, specifically PLA2s and LPATs, can regulate membrane tubule formation by controlling the lipid composition on one-half of a lipid bilayer.
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Materials and Methods |
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The following antibodies were generously supplied to us: rabbit polyclonal anti--mannosidase II (Kelly Moremen, University of Georgia); mouse monoclonal anti-GS28 (Dr Wanjin Hong, Institute of Molecular and Cell Biology, Singapore); and rabbit polyclonal anti-GPP130 (Adam Linstedt, Carnegie Mellon University). Mouse monoclonal anti-TGN38 antibody was purchased from Affinity BioReagents (Golden, CO). All secondary fluorescent antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Jennifer Lippincott-Schwartz (NICHD, Bethesda, MD) kindly provided the expression vector that encodes a cis Golgi-restricted mutant form of the KDEL-R coupled to GFP (Sciaky et al., 1997
). Mouse monoclonal anti-transferrin receptor (TfR) antibody was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Marino Zerial kindly provided the expression vector that encodes Rab11 coupled to GFP (Sonnichsen et al., 2000
).
Cell culture, transfection and fluorescence microscopy
Clone 9 rat hepatocytes and HeLa cells were maintained in minimal essential medium (MEM) with 10% Nu-Serum and 1% penicillin/streptomycin at 37°C in an atmosphere of 95% air and 5% CO2. For some experiments, Clone 9 cells were transfected with CLONfectin from CLONTECH (Palo Alto, CA) as described by the manufacturer. Clone 9 cells were grown on glass coverslips for 2 days before experiments were performed. Cells were washed three times with serum-free MEM and incubated in serum-free MEM containing inhibitors at the concentrations and for the times indicated in the Results. In previous studies, we found that the IC50 of CI-976 for a rat liver Golgi membrane-associated LPAT is 15 µM and that at 50 µM the activity was completely inhibited (Drecktrah et al., 2003
). Also, treatment of cells with CI-976 between 20-50 µM induced reversible Golgi tubulation. Therefore, for experiments here we used concentrations between 20-50 µM. We note that CI-976 was not active in media containing serum.
For immunofluorescence microscopy, cells were fixed with either 3.7% formaldehyde or 100% methanol at 20°C (for TGN38). Primary antibodies used were: mouse anti-GS28 diluted 1:100, mouse 10E6 diluted 1:100, rabbit anti-GPP130 diluted 1:500, rabbit anti-ManII diluted 1:1000, mouse anti-TGN38 diluted 1:100, rabbit anti-M6PR diluted 1:2000. Secondary antibodies used were: goat anti-mouse FITC diluted 1:100, donkey anti-rabbit TRITC diluted 1:100, and goat anti-mouse rhodamine diluted 1:100. When staining for TGN38, 1% BSA was added to the primary and secondary antibodies. For most experiments, cells were viewed by wide-field epifluorescence (Zeiss Axioskop 2). In other experiments, cells were examined with a Perkin-Elmer UltraView spinning disc confocal microscope.
For CI-976 dose-response experiments on Tf recycling, HeLa cells were plated on glass coverslips 2 days prior to the experiment. Cells were briefly washed in MEM minus serum and then incubated for 45 minutes in the presence of Alexa568-Tf. Following the transferrin uptake, cells were washed as above and incubated in various concentrations of CI-976 for 1 hour. Cells were then fixed and processed for immunofluorescence as above. To quantify the effects of CI-976 on the recycling of Tf, cells were imaged and classified for brightness using Openlab (Improvision, Lexington, MA) software. Using the Region of Interest (ROI function), a region was drawn around the perimeter of each cell (a minimum of 30 cells per experiment) and the mean pixel intensity was calculated. To correct for background fluorescence a ROI of an area devoid of cells was measured for pixel intensity. The background pixel intensity was then subtracted from all of the means to give accurate pixel intensity.
For the live cell imaging of endosome tubulation induced by CI-976, HeLa cells stably expressing galactosyltransferase-GFP (HeLa-GalT-GFP) (Storrie et al., 1998) cells were used. Cells were allowed to endocytose Alexa568-Tf for 45 minutes, washed to remove unbound transferrin, and then placed in media (MEM, 2.6 mM sodium bicarbonate, 5 mM HEPES) containing 25 µM CI-976 and imaged immediately on the spinning disc confocal microscope.
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Results |
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We first examined the cis Golgi (and CGN) using well-characterized antibodies or GFP constructs of markers found primarily in this region: GPP130 (Puri et al., 2002), GS28 (Subramaniam et al., 1996
), a mutant form of the KDEL-R coupled to GFP (Sciaky et al., 1997
), and the 10E6 antigen (Wood et al., 1991
). With the exception of 10E6, all were found in tubules extending from the juxtanuclear Golgi region after short incubations (10-15 minutes) in CI-976 (Fig. 1B,E). By 20-30 minutes, all of the cis Golgi markers, including 10E6, started to become diffuse in the cytoplasm in an ER-like pattern. After extended periods of time (>50 minutes), a large proportion of the cells had this diffuse staining pattern (Fig. 1C,F). The medial marker, ManII, behaved similar to the cis markers (Fig. 1G-I). Two markers of the trans Golgi network (TGN), TGN38 and the mannose 6-phosphate receptor (M6PR), were also found in tubules following short-term treatment (10-15 minutes) with CI-976; however, these markers were not found diffuse in the cytoplasm after extended periods of time (>50 minutes) (Fig. 1J-O). Instead, they assumed a juxtanuclear distribution, similar but not identical to control cells in that stained elements were more tightly clustered following long-term CI-976 treatment (Fig. 1L,O). This redistribution of TGN was different than cis, medial or the trans markers, all of which assumed a diffuse ER-like pattern after an extended period of time in CI-976. To determine which subcompartment was most responsive to CI-976, we counted the number of cells with tubules as revealed by each maker antibody. These studies were complicated by the fact that some antibodies produce brighter signals than do others. However, the use of multiple antibodies against cis or trans compartments provided a measure of confidence in the results that suggested the following order of responsiveness to CI-976: medial
trans>cis. In addition, the ability of CI-976 also to induce tubule formation in HeLa cells (data not shown) indicates that this effect is not cell-type specific.
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TGN and early endosomes do not fuse
As noted above, the two TGN markers, TGN38 and M6PR, were found in tubules following short-term treatment with CI-976, but after extended periods of time they were more tightly clustered. This response is similar to that seen in BFA-treated cells, in which the TGN fuses with the early endosomes (Lippincott-Schwartz et al., 1991; Wood et al., 1991
). To determine whether the TGN was behaving similarly in the presence of CI-976, cells were treated with 20 µM CI-976 for 50 minutes and then labeled with Alexa568-dextran for 5 minutes, while still in CI-976, to allow endocytic delivery to early endosomes. M6PRs served as a marker for the TGN and its fusion with early endosomes as demonstrated in previous studies with BFA (Wood and Brown, 1992
; Wood et al., 1991
). The results of confocal imaging showed that in control (Fig. 3A1-A3) and CI-976-treated cells (Fig. 3B1-B3), Alexa568-dextran internalized for 5 minutes did not co-localize with M6PRs to any significant extent. We conclude that the TGN and early endosomes do not fuse in the presence of CI-976.
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CI-976 does not inhibit initial uptake of Tf
Because CI-976 did not cause the TGN to fuse with endosomes but did cause endosomes to form tubules, we wondered if CI-976 might also be affecting endocytic compartments and trafficking. To investigate this issue, the well-characterized intracellular trafficking of TfR and its ligand Tf were followed (Maxfield and McGraw, 2004). First we assessed whether CI-976 affected initial uptake of Tf. TRITC-Tf was bound to Clone 9 cells for 2 hours at 4°C, and the unbound TRITC-Tf was removed by extensive washing. Cells were then treated for 15 minutes with 20 µM CI-976 (or DMSO as a solvent control) at 4°C prior to shifting to 37°C for 5 minutes to allow TRITC-Tf uptake. Treatment with CI-976 did not prevent uptake of TRITC-Tf from the cell surface (Fig. 5).
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This compact juxtanuclear staining pattern suggests that CI-976 was inhibiting the recycling of Tf. To examine this further, and to establish that the CI-976 effect is not cell-type specific, a series of dose-response experiments on Tf recycling were quantified using HeLa cells. As shown in Fig. 7, it is clear that CI-976 inhibits recycling at >2 µM and that the effect becomes more pronounced up to 50 µM. These experiments could only be done up to 50 µM CI-976 because cells became unhealthy at higher concentrations. These results suggest that CI-976 inhibited the exit of Tf from the central endocytic recycling compartment.
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To determine if TfRs also accumulate in this juxtanuclear cluster, HeLa cells were treated as described immediately above and then stained for immunofluorescence localization of TfR. In control cells, Tf and TfR were co-localized in both peripheral sorting and central recycling endosomes (Fig. 8A,B). In the presence of CI-976, Tf and TfR were found to co-localize in a compact, juxtanuclear staining pattern (Fig. 8C,D), suggesting that recycling of TfRs in these cells is similarly inhibited by CI-976.
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To see if other endocytic trafficking pathways were disrupted by CI-976, we examined the uptake and delivery of LDL via the endocytic degradation pathway to lysosomes. In these experiments, fluorescently labeled DiI-LDL and Alexa488-EGF were followed in pulse-chase experiments, and we found that CI-976 did not detectably inhibit the transport of LDL or EGF from peripheral early sorting endosomes to larger, centrally located late endosomes or lysosomes (Fig. 10). These results show that CI-976 had a selective effect on trafficking in the endocytic recycling pathway.
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Tf accumulates in Rab11-positive recycling endosomes
The central location of Tf/TfRs in CI-976-treated cells suggests that the endocytic recycling compartment is the site of export blockage (Maxfield and McGraw, 2004). To examine this further, Clone 9 cells were transiently transfected with a GFP-tagged Rab11 construct, a known marker of the central endocytic recycling compartment (Sonnichsen et al., 2000
). The cells were then pretreated for 10 minutes with 20 µM CI-976 prior to labeling with Alexa568-Tf for 45 minutes in the continued presence of CI-976. The cells were visualized by confocal microscopy. Images shown are three consecutive slices 0.3 µm apart. In control cells in which Tf is delivered to all endocytic compartments, both individual and merged images show that Tf and Rab11 co-localize slightly (Fig. 11A1-A3). However, when cells were treated with CI-976, Tf and Rab11 had a high level of co-localization in a tight juxtanuclear staining pattern (Fig. 11B1-B3). This result shows that the Tf-TfR complexes accumulate in the central endocytic recycling compartment. Interestingly, in CI-976 treated cells, GFP-Rab11 also became more concentrated in a tight juxtanuclear position, suggesting that its cycling is also affected by CI-976. Although Tf-TfR accumulated in the compact endocytic recycling compartment following CI-976 treatment, electron microscopy revealed no obvious differences in the morphology of the compartment (data not shown).
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Tf recycling is irreversibly inhibited by CI-976
To determine if the effect of CI-976 on the Tf trafficking pathway was reversible, cells were pretreated with CI-976 for 10 minutes and then pulse-labeled with TRITC-Tf for 45 minutes to accumulate Tf in the endocytic recycling compartment. Cells were then extensively washed and chased in medium without CI-976. As above, in control cells with no chase, TRITC-Tf was found in peripheral and central endosomes (Fig. 12A), whereas in CI-976 treated cells TRITC-Tf was found in a tight juxtanuclear staining pattern (Fig. 12C). As expected in control cells following a 1 hour chase, TRITC-Tf staining was significantly reduced, consistent with its recycling and loss into the extracellular medium (Fig. 12B). However quite different results were obtained with CI-976 treated cells. Following a 1 hour chase in which CI-976 had been removed, TRITC-Tf was not lost from cells but instead remained in a tight juxtanuclear staining pattern (Fig. 12D). Interestingly, even when the chase was extended to 24 hours, TRITC-Tf remained in the juxtanuclear cluster (Fig. 12E). Similar experiments on HeLa cells showed that although Tf did not chase from the cells after 24 hours, Tf-positive vesicles were somewhat more diffuse throughout the cytoplasm, when compared to Clone 9 cells. Although recovery of TfR recycling was extremely slow, cell viability was not significantly affected and cells continued to grow indicating that recovery of recycling eventually occurs. These results suggest that the molecular target of CI-976 in the endocytic recycling pathway is very slowly reversible, and perhaps even irreversible. The slow recovery seen could be due to new protein synthesis.
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Discussion |
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We have previously shown that CI-976 inhibits a novel Golgi-associated LPAT and that this compound stimulates tubule formation and retrograde trafficking to the ER (Drecktrah et al., 2003). Here we show that CI-976, similar to BFA, is capable of inducing tubule formation from all regions of the Golgi complex, including the TGN, and endosomes. However, whereas BFA induces tubule-mediated retrograde trafficking of Golgi membranes to the ER and fusion of the TGN with early endosomes (Lippincott-Schwartz et al., 1991
; Wood et al., 1991
), CI-976 induced only the former. In addition, CI-976 inhibited export of Tf/TfRs from a Rab11-positive recycling endosome compartment, whereas endocytic recycling is often stimulated by BFA (Lippincott-Schwartz et al., 1991
; Wood et al., 1991
). Interestingly, reassembly of the Golgi complex following recovery from CI-976 treatment was fairly rapid, whereas inhibition of export from recycling endosomes was only very slowly reversible. These results strongly suggest that the molecular targets of CI-976 are different in the Golgi complex and endosomes.
The idea that membrane curvature and function is controlled by enzyme-catalyzed PL/LPL conversion has recently been gaining support (Brown et al., 2003; Burger, 2000
; Huijbregts et al., 2000
; Huttner and Schmidt, 2002
; Shemesh et al., 2003
), although no exact enzymes have yet been identified. Our previous studies have shown that PLA2 antagonists are potent inhibitors of membrane tubule formation (de Figueiredo et al., 1998
; de Figueiredo et al., 2000
; de Figueiredo et al., 1999
; Drecktrah and Brown, 1999
; Polizotto et al., 1999
). These results led us to propose that cytoplasmic PLA2 enzyme(s) may act to generate localized increases in inverted cone-shaped LPLs, thus contributing to an increase in outward membrane bending and tubule formation (Brown et al., 2003
; de Figueiredo et al., 1998
). By inhibiting the reacylation of these LPLs with CI-976, a similar increase in membrane LPL concentration results (Drecktrah et al., 2003
), leading to the formation of tubules seen here.
In addition to increasing outward curvature and tubulation via the accumulation of LPC, CI-976 might also inhibit membrane trafficking by inhibiting the formation of inward curve inducing lipids, such as phosphatidic acid (PA) or diacylglycerol (DAG), that have been implicated in vesicle formation. For example, endophilins A and B and CtBP/BARS are LPAATs that catalyze the conversion of LPA to PA and have been implicated in membrane fission (Scales and Scheller, 1999; Schmidt et al., 1999
; Weigert et al., 1999
). In these cases, re-acylation of inverted cone-shaped LPA back to cylindrical or cone-shaped PA might contribute to inward bending that occurs at the neck of a budding vesicle (Burger, 2000
; Huijbregts et al., 2000
; Shemesh et al., 2003
). PA could also be metabolized to DAG by phospholipase D, an enzyme implicated in coated vesicle formation including most recently, COPII vesicles (Bi et al., 1997
; Pathre et al., 2003
; Roth et al., 1999
). Interestingly, one known target of BFA is CtBP/BARS (Spano et al., 1999
). Since BFA and CI-976 have similar effects on the Golgi complex, CtBP/BARS may be a possible target of CI-976. However, CtBP/BARS is an LPA-specific LPAAT, whereas CI-976 was found to be selective for a Golgi-associated LPCAT (Chambers and Brown, 2004
; Drecktrah et al., 2003
). In addition, CtBP/BARS is capable of inducing fission of membrane tubules when its LPAAT activity is compromised (Carcedo et al., 2004
). Thus, the Golgi target of CI-976 cannot be CtBP/BARS.
Although CI-976 does not appear to influence the activity of a Golgi LPAAT, it could affect unknown LPAATs involved in vesicle production from endosomes, thus accounting for its ability to inhibit export of Tf/TfRs from recycling endosomes. Many studies have provided evidence that coated vesicles may work in concert with membrane tubules to facilitate export from endosomes (Bonifacino and Glick, 2004; Bonifacino and Lippincott-Schwartz, 2003
). In other words, CI-976 could inhibit different acyltransferases that influence membrane trafficking by two specific mechanisms. First, failure to reacylate LPLs generated by PLA2 activity on Golgi membranes would stimulate tubule formation leading to inappropriate retrograde trafficking to the ER. Second, CI-976 could inhibit an unknown LPAAT involved in membrane fission and vesicle production from recycling endosomes, thus accounting for the inhibition of Tf recycling. Inhibition of vesicle fission by CI-976 might also explain why relocated Golgi enzymes fail to exit the ER (Drecktrah et al., 2003
). Thus, it is tempting to speculate that CI-976 might be a fission inhibitor specific for some (COPII?), but not all (e.g. AP-2 clathrin coated vesicles) vesiculation events.
If the effects of CI-976 are indeed due to the inhibition of an LPAT, then prior treatment with a PLA2 antagonist, to prevent the formation of LPLs, should abrogate or reverse the effects of CI-976. In support of this idea, pretreatment of cells with PLA2 antagonists prior to CI-976 inhibits Golgi membrane tubule formation and retrograde trafficking to the ER (Drecktrah et al., 2003). Thus, one might expect that PLA2 antagonists would reverse the inhibitory effects of CI-976 on Tf recycling. However, that experiment cannot be done in the context of endocytic recycling because we have previously shown that PLA2 antagonists on their own inhibit both endosome tubule formation and Tf recycling (de Figueiredo et al., 2001
). Taken together, the data strongly suggest that recycling endosomes utilize a two-step process for efficient export and recycling: PLA2-facilitated tubule formation followed by LPAAT-facilitated membrane fission to generate vesicles. In this regard it is worth noting that CI-976 does not cause COPI proteins or ADP-ribosylation factor to dissociate from the Golgi complex (Drecktrah et al., 2003
); therefore, its proposed effects on vesiculation are consistent with inhibition of processes that follow coat protein binding, i.e. fission.
In conclusion, like BFA, CI-976 has been shown to be a versatile tool for studying membrane trafficking events in both the secretory and endocytic pathways. Owing to differences in the effects of BFA and CI-976 (particularly on the endocytic pathway), the mechanisms of action on these two compounds are most certainly very different. Here we propose that CI-976 inhibits multiple acyltransferases involved in various steps of the secretory and endocytic pathways, which is consistent with recent proposals that acyltransferases influence membrane shape and function by regulating PL and LPL levels.
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Acknowledgments |
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