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Vol. 18, Issue 11, 4637-4647, November 2007
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*Centre National de la Recherche Scientifique Unité Mixte de Recherche 144, 75248 Paris Cedex 05, France; Institut Curie, 75248 Paris Cedex 05, France; and Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-5430
Submitted August 19, 2007;
Accepted August 31, 2007
Monitoring Editor: Sandra Schmid
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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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). During interphase, organelle damage, intracellular remodeling, or simply cellular activity also necessitates a precise control of organelle dynamics. How the organelles are built, maintained, and degraded thus represent key questions that remain unsolved for most organelles.
One particularly studied example is the dynamics of the Golgi apparatus. The Golgi complex play a central role in eukaryotic cell homeostasis. It processes and sorts proteins and lipids synthesized in the endoplasmic reticulum (ER) and serves as a central platform connecting the anterograde and retrograde trafficking pathways. These activities are coupled to unique ultrastructural characteristics. The Golgi apparatus is composed of stacks of flattened, adherent cisternae (Rambourg and Clermont, 1997; Ladinsky et al., 1999). In certain eukaryotes, and in particular in humans, hundreds of stacks are laterally connected to form an extended ribbon-like structure next to the microtubule-organizing center (MTOC). Each stack displays an internal polarity: the cis side exchanges material from the ER through the intermediate compartment and the cis-Golgi network; at the other extremity, the trans-Golgi, separated from the cis-Golgi by intervening medial cisternae, is in contact with the trans-Golgi network (TGN). The TGN executes final sorting steps to target multiple post-Golgi destinations and exchanges material with the endocytic pathway.
Despite the large and continuous flow of membranes and proteins occurring at steady state, the overall organization, ultrastructural shape, and polarity of the Golgi apparatus is remarkably stable. Each Golgi cisternae contains a particular set of "resident" proteins, such as glycosylation enzymes, but how this is maintained is still debated (Martinez-Menarguez et al., 2001; Cosson et al., 2002; Altan-Bonnet et al., 2004; Puthenveedu and Linstedt, 2005; Storrie, 2005). Two extreme models have been proposed to explain how such a structure is dynamically maintained. According to the "static cisternae" model, cargo advances, packed in vesicles or using extended tubules, through a stable stacked structure (Pelham, 2001). According to this model, resident proteins are stably localized to particular cisternae using specific signals, through interactions with the membranes or with the matrix, a sort of stable and inheritable exoskeleton that may serve as a template for the maintenance of the Golgi complex. This matrix is particularly important upon mitosis exit (Shorter and Warren, 2002) but is also proposed to play a role during interphase to maintain Golgi structure (reviewed in Glick, 2002). According to the "cisternal maturation" model, the Golgi apparatus in endowed with auto-organization abilities and does not depend on an external matrix to build and maintain its structure. Cargo are transported inside maturating cisternae and resident proteins achieve their steady-state localization through retrograde transportation (reviewed in Glick, 2002; Shorter and Warren, 2002; Barr, 2004). Independently of the model, intercisternae transport (respectively of cargo or of resident proteins) may occur via vesicular or tubular connections (for reviews see Mironov et al., 2005; Rabouille and Klumperman, 2005).
Recent studies strongly support the model of cisternal maturation for yeast Saccharomyces cerevisiae (Losev et al., 2006; Matsuura-Tokita et al., 2006). In higher eukaryotes, many studies have tried to address the question of Golgi apparatus dynamics using in vivo imaging of green fluorescent protein (GFP)-tagged reporters, recovery from drug treatments, or Golgi re-formation after mitosis (Shima et al., 1997; Zaal et al., 1999; Jokitalo et al., 2001; Puri and Linstedt, 2003; Axelsson and Warren, 2004; Nizak et al., 2004; Pecot and Malhotra, 2004). Few studies, however, have directly addressed the question of Golgi apparatus maintenance in mammalian interphase cells (see however, Pecot and Malhotra, 2006). One way to study it would be to remove the Golgi from living cells and study recovery. Such an idea was followed using cytoplast generated by microsurgical methods (Pelletier et al., 2000). This led to the observation that no Golgi can re-form in cytoplast containing only peripheral ER. However, this method is a cell by cell approach, assumes that no Golgi exist in the cell periphery, and above all is technically demanding and has so far only been mastered by one team (Pelletier et al., 2000; Sheff et al., 2002).
Here we develop a new method that allows one to enzymatically inactivate the Golgi apparatus in intact living cells. We took advantage of two sets of studies, both centered on the use of horseradish peroxidase (HRP). On the one hand, HRP has been used to inactivate the function of endosomes (Futter et al., 1996; Brachet et al., 1999; Pond and Watts, 1999). In these studies, endosomes are filled with HRP, alone or fused to transferrin or to EGF, and treated with 3,3'-diaminobenzidine (DAB) in the presence of a low concentration of H2O2. HRP-dependent polymerization of DAB cross-links the target organelle and blocks its dynamics. This treatment is specific for organelles loaded with HRP, here the endosomes, and does not affect the function of other organelles like the Golgi (Brachet et al., 1999). On the other hand, the work of Cutler, Hopkins, and coworkers showed that active HRP can be expressed in the lumen of secretory compartments and, when fused to the transmembrane domain of the trans-Golgi/TGN enzyme sialytransferase, HRP can be stably expressed in the lumen of Golgi cisternae (Connolly et al., 1994; Stinchcombe et al., 1995).
We devised ways to apply HRP-based organelle cross-link to the Golgi apparatus and analyze whether it allows specific and drug-free inactivation of Golgi dynamics and function, providing a unique set-up to study Golgi maintenance in interphase cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), anti-GMAP210 was kindly provided by Dr. M. Bornens (Institut Curie, Paris, France), anti-GM130 was from BD Biosciences (San Jose, CA), and anti-TGN46 was from Serotec (Raleigh, NC).
Plasmid Constructs
pManII-HRP was constructed by replacing the AgeI/NotI enhanced GFP (EGFP) insert from pManII-EGFP plasmid (kind gift from J. White, EMBL, Germany) by the HRP coding sequence. HRP was amplified by PCR using the following primers: 5' CGCGGATCCACCGGTCGCCACC ATG CAG TTA ACC CCT ACA TTC 3' and 5' TACCTAGGTGCGGCCGCTTAAGAGTTGCTGTTGACCAC 3', cut with AgeI and NotI and inserted in the target plasmids opened similarly.
Cell Culture
HeLa cells were grown in DMEM containing 4.5 g/l glucose supplemented with 10% fetal calf serum, penicillin-streptomycin, and sodium pyruvate in a 5% humidified CO2 incubator. Transfection was achieved using the calcium phosphate precipitate method described in Jordan et al. (1996). Stable cell lines expressing mannosidase II (ManII)-HRP were obtained after selection using 400 µg/ml geneticin.
For transferrin uptake, 5 µg/ml Alexa633-labeled transferrin was added in the culture medium at the same time as BFA, and cells were incubated for 90 min at 37°C.
Golgi cross-linking was achieved essentially as described in Brachet et al. (1999) for endosome cross-linking after HRP internalization. Cells expressing or not HRP in Golgi cisternae were washed in PBS and incubated with DAB + 0.003% H2O2 for 30 min at 0°C. Cells were then washed and fixed or further incubated in normal medium before fixation.
Electron Microscopy
Epon Embedding.
DAB labeling was either done on living cells as described above or on fixed cells as previously (Nizak et al., 2004) using DAB (0.25 mg/ml, Sigma) and 0.003% H2O2 (Sigma) in phosphate-buffered solution for 40 min. Cells were then fixed in 4% paraformaldehyde and 0.5% glutaraldehyde for 4 h at room temperature, rinsed, and postfixed 1 h at room temperature in reduced osmium (1:1 mixture of 2% aqueous potassium ferrocyanide) as described previously by Karnovsky (1971).
After postfixation the cells were embedded in agar 2%, dehydrated in ethanol, and processed for Epon embedding. Thin sections were cut on a Reichter-E ultramicrotome (Reichter-Jung, Vienna, Austria), collected on copper grids and stained with lead citrate for 2 min. Sections were then examined with a CM 10 Philips electron microscope (FEI, Eindoven, The Netherlands) at 80 kV.
Immunogold Labeling.
Transfected HeLa cells were incubated with DAB + H2O2 on ice for 30 min and fixed with 2% (wt/vol) paraformaldehyde with 0.2% (wt/vol) glutaraldehyde, in 0.1 M phosphate buffer, pH 7.4. Cell pellets were processed for ultracryomicrotomy as previously described (Raposo et al., 1997). Ultrathin cryosections were prepared with an ultracryomicrotome Ultracut FCS (Leica, Vienna, Austria) and immunogold labeled with the indicated primary antibodies and using protein A conjugated to 10-nm gold (Cell Microscopy Center, AZU, Utrecht, The Netherlands). Sections were analyzed under a Philips CM120 electron microscope, and digital acquisitions were made with a numeric camera Keen View (Soft Imaging Systems, Münster, Germany).
Photoconversion
ManII-HRP cells were transfected with a plasmid controlling the expression of Dendra2-GM130 (Evrogen, Moscow, Russia). Twenty-four hours later, cells were observed alive in complete medium using a Ludin chamber placed on a LSM510-confocal microscope and imaged sequentially using green and red fluorescence settings. Cells were treated with DAB/H2O2 for 30 min on ice, washed, warmed-up in complete medium at 37°C, and imaged in green and red channels. Dendra2-GM130 located on the Golgi apparatus were then specifically photoconverted using a 405-nm laser beam scanning in a region of interest to avoid photoconversion of the cytoplasmic form of GM130. Cells were imaged just after the photoconversion and 90 min later. A transmitted light image was also acquired to localize the cross-linked, DAB-positive Golgi apparatus.
Vesicular Stomatitis Virus G Protein Trafficking
Cells were transfected with a plasmid allowing the expression of GFP- VSVGts045 and incubated overnight at 40°C. Transport to the Golgi complex and to the plasma membrane was initiated by shifting cells at 32°C. In certain experiments, cells were cooled down at 0°C and cross-linked using DAB/H2O2 before shifting at 32°C. Expression at the plasma membrane was quantified using the VG antibody, directed against the exoplasmic domain of vesicular stomatitis virus G (VSV-G) protein, in the absence of detergent. After incubating the cells with a fluorescent anti-mouse antibody, when necessary cells were rapidly fixed again using paraformaldehyde, permeabilized, and counterstained using another antibody. For fluorescence-activated cell sorting (FACS) analysis, cells were first trypsinized before being fixed and stained. For quantification of Golgi arrival, sensitivity of VSV-G glycosylation to EndoH, cells expressing GFP-VSV-Gts045 blocked in the ER overnight at 40°C were pulse labeled using 150 µCi/ml [35S]TransLabel (ICN Biomedicals), washed in radioactive-free medium, cross-linked or not, and incubated at 32°C. Cells were then lysed in immunoprecipitation (IP) buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 5 mM EDTA, 1% Triton X-100, and 0.20% bovine serum albumin [BSA]), and GFP-VSVG was precipitated using monoclonal anti-GFP, and treated or not with EndoH following the instructions of the provider, and radioactive proteins were separated by SDS-PAGE and quantitatively imaged using a PhosphorImager (GE Healthcare, Waukesha, WI).
Fluorescence Microscopy
Cells were stained using Alexa488-labeled (Molecular Probes) or Cye-dye–labeled secondary antibodies (Jackson ImmunoResearch). Alternatively the natural fluorescence of GFP and Dendra2 was used. Optical microscope used in this study were as follows: 1) Leica DMRA (Wetzlar, Germany) equipped with a Micromax cooled CCD camera controlled by the Metamorph software (Molecular Devices, Berkshire, United Kingdom). Images were acquired using the 63x NA 1.32 oil ph3 CS (HCX PL APO) objective, and fluorescence filters A4, L5, Y3, and Y5 from Leica; and 2) Zeiss LSM510 meta installed with a 405-nm laser diode and argon and helium laser (Thornwood, NY). The objective used for imaging was the 63x NA 1.4 oil differential interference contrast (DIC; Plan Apochromat) together with the fluorescein isothiocyanate (FITC) and Rhod filters.
Deconvolution and Three-dimensional Image Processing
Cells fluorescently labeled were examined under a classical upright motorized microscope (Leica DMRA2). Images were acquired using an oil-immersion objective (x100 PL APO HCX, 1.4 NA) mounted on a piezo-electric motor (LVDT, Physik Instruments, Waldbronn, Germany) and a high-sensitive cooled interlined CCD camera (Roper CoolSnap HQ, Tucson, AZ). The system was controlled by the Metamorph Software (Molecular Devices). Deconvolutions were performed on stacks of images taken with a 0.3-µm plane-to-plane distance, using the three-dimensional (3D) deconvolution module of Metamorph and the fast iterative constrained Point Spread Function-based algorithm. Surface rendering and volume quantifications were performed using Amira software (TGS, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, although usable, also stained post-Golgi elements after DAB treatment. We thus constructed Golgi-targeted HRP-fusion proteins by fusing HRP to the transmembrane domains of different Golgi enzymes. This strategy has been successfully used before to target GFP to specific Golgi subcompartments (Storrie et al., 1998). We removed the enhanced GFP (EGFP) from pManII-EGFP and replaced it with HRP (see Materials and Methods). Immunofluorescence experiments showed that, as its fluorescent counterpart, ManII-HRP stably expressed in HeLa cells and stained using an anti-HRP antibody was colocalized with Golgi markers like GM130 or TGN46 (Figure 1A). The experiments that we report here were done using a stable ManII-HRP HeLa clone. We further confirmed that, similar to ST-HRP (Connolly et al., 1994), these fusion proteins allow efficient targeting of active HRP to Golgi cisternae. Golgi localized HRP activity was revealed on fixed cells by DAB/H2O2, and the DAB precipitate colocalized with cis to trans-Golgi markers like GM130 and TGN46.
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to achieve ablation of early endosomes after endocytosis of transferrin-HRP, because very little nonspecific effects of DAB/H2O2 were observed in their conditions. HeLa ManII-HRP were thus treated with diluted DAB in the presence of a low concentration of H2O2 for 30 min on ice. Similar results were obtained after 15 min at room temperature. This treatment led to the formation of a brown precipitate in the region of the Golgi. Immunofluorescence experiments using different Golgi markers like GM130 and TGN46 (Figure 1B, a–c) confirmed that this precipitate indeed stained the Golgi complex. No such precipitate was observed after treatment of living control HeLa cells (Figure 1B, d and e). We noticed that antibody staining of certain luminal markers, such as endogenous galT or exogenous ManII-HRP, is strongly reduced after DAB/H2O2 treatment as if antigen accessibility was compromised due to DAB precipitation. This possibility was further suggested by the fact that the signal corresponding to fluorescent proteins like EGFP-galT was not affected to the same extent. We further confirmed that the DAB precipitate was specifically formed in the lumen of Golgi cisternae by electron microscopy (EM). ManII-HRP cells were fixed and incubated with DAB/H2O2 before being processed for EM. The DAB precipitate was deposited in either the medial and trans cisternae, or in all Golgi cisternae, probably depending on the expression level (Figure 2A). No staining was detected in the ER or in wild-type HeLa cells (Figure 2C). Importantly, a Golgi staining was also observed in cells incubated before fixation with DAB/H2O2 (Figure 2B), indicating that the DAB precipitate is specifically formed in the lumen of Golgi cisternae in living cells.
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Perturbation of Golgi Dynamics after Cross-Linking
According to the endosome ablation studies and because the DAB precipitate was only found inside the Golgi cisternae, it was likely that DAB/H2O2 treatment would lead to the specific cross-linking and hence inactivation of the Golgi complex. We tested this hypothesis by evaluating the sensitivity of organelles in cross-linked cells to particular drug treatments. ManII-HRP cells were incubated or not with DAB/H2O2 before being treated by BFA. BFA addition leads to the rapid relocation of Golgi markers such as Golgi enzymes or giantin to the ER, whereas others like GM130 or GMAP210 are transported into the intermediate compartment, mixed with the endosomes (e.g., TGN46), or released to the cytosol (e.g., COPI). This illustrates the extreme dynamics that underlies steady-state Golgi localization. In nontreated cells (Figure 3A, a–c), we confirmed that BFA treatment induces the relocation of giantin to the ER, whereas GM130 is transported to the intermediate compartment. Addition of BFA also induces a strong tubulation of early endosomes, as observed by fluorescent transferrin endocytosis (Figure 3A c). In contrast, after Golgi cross-linking (Figure 3A, d–f), ManII-HRP cells did not react to BFA by relocating Golgi markers. Careful examination revealed only weak GM130 in the cell periphery after BFA treatment but the majority of GM130 and giantin staining stays associated to the DAB-positive Golgi. One possibility was that DAB/H2O2 incubation of ManII-HRP cells was toxic, inactivating nonspecifically membrane dynamics, or that BFA sensitivity was lost after DAB/H2O2 treatment. In cross-linked cells, however, early endosomes still became tubular and continued to efficiently uptake fluorescent transferrin (Figure 3Af). Thus, cross-linked cells are still alive and dynamic and BFA is still active in these cells. This experiment confirms that, as shown before (Futter et al., 1996; Brachet et al., 1999; Pond and Watts, 1999), DAB/H2O2 treatment did not generally affect the cells and indicates that the Golgi complex is no longer dynamic after DAB/H2O2-based cross-linking.
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; Storrie et al., 1998). Because this dispersion is strongly dependent on an active Golgi-to-ER pathway (Storrie et al., 1998), we tested whether cross-linked Golgi still dispersed in the presence of nocodazole. Non-DAB/H2O2–treated ManII-HRP cells reacted normally to nocodazole addition, and a dispersed Golgi was observed after a 90-min treatment at 37°C as imaged by anti-GM130 and giantin antibodies (Figure 3B, a and b). In contrast, very little Golgi dispersion was observed after nocodazole addition in ManII-HRP cells pretreated with DAB/H2O2 (Figure 3B, d–f). In these cells, the shape of giantin- and GM130-positive Golgi membranes was almost unaffected by nocodazole treatment, and these membranes stayed colocalized with the DAB precipitate. This further indicated that DAB precipitation inactivates Golgi dynamics.
In Vivo Cross-Linking of ManII-HRP–positive Compartment Leads to Golgi Function Inactivation
We next tested whether incubation of ManII-HRP cells with DAB/H2O2 led to inactivation of the Golgi apparatus function. Secretion was quantified using the ts045 mutant of the VSV-G protein. This protein remains in the ER at restrictive temperature (40°C) and only folds properly, trimerizes, and exits the ER at permissive temperature (32°C). At early time points, the VSV-G protein transits through the Golgi complex (30 min), where its glycosylation becomes Endo H– resistant and reaches the plasma membrane later on (after 45–60 min). The use of the VG mAb directed against the exoplasmic domain of VSV-G allows one to quantify, using nonpermeabilized cells, the extent of cell surface expression. The total amount of VSV-G is either visualized using the monoclonal P5D4 (when using viral infection) or the fluorescence of the recombinant VSVG-EGFP fusion molecule.
We first analyzed VSV-G trafficking by immunofluorescence and microscopic observation (Figure 4A). Control HeLa ManII-HRP cells treated only with H2O2 secreted VSV-G with normal kinetics: it was accumulated in the ER at 0 min and then was transported toward the Golgi apparatus (30 min) before being expressed at the plasma membrane (Figure 4A, a–d). In contrast, only low plasma membrane staining was observed in HeLa ManII-HRP treated with DAB/H2O2 (Figure 4A, e–h), indicating a strong decrease in protein VSV-G secretion after cross-linking. This block was quantified by FACS analysis (Figure 4B).
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Golgi Complex Rebuilding after Inactivation
The in vivo cross-linking of ManII-HRP leads to ablation of Golgi function. A key question is whether a new Golgi apparatus can re-form after inactivation or whether the cell needs a pre-existing Golgi structure to build a new Golgi. We thus studied Golgi recovery after cross-linking, following the behavior of several Golgi markers: transmembrane proteins located in the cis- and medial Golgi (giantin) and in the trans/TGN (TGN46) as well as peripheral proteins (GM130). As expected, immunofluorescence staining of treated cells showed that just after Golgi inactivation (Figure 5A, a and e), these three proteins were detected in largely overlapping Golgi stained by DAB. As soon as 30 min after the cross-link, GM130 was detected on new structures, often found apposed to the "old" inactivated Golgi, whereas the two transmembrane proteins, giantin and TGN46, were still located only on DAB-positive Golgi elements (Figure 5A, b and f). At later time points, more GM130 was detected on these new structures and was progressively lost by the old inactivated ones. The proportion of TGN46 detected on the GM130-positive new structures also increased but to a lesser extent (Figure 5A, c and d). In contrast, giantin did not partition at all on these elements and was still found located on the inactivated Golgi. 3D reconstruction after deconvolution confirmed that, whereas in control cells GM130 and giantin are closely apposed on Golgi structures (Figure 5Ba), after cross-linking they partition on two separate structures (Figure 5Bb).
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) and the Golgi v-SNARE GS15 (Xu et al., 1997, 2002). Surprisingly, although GS15 was not found on newly formed Golgi-like elements, its partners YKT6, GS18, and syntaxin-5 (Xu et al., 2002) were localized on these new structures. Note also that the Golgi enzyme galactosyl transferase was absent from re-formed structures.
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). To answer to this question, we set up a photo-conversion experiment that allows to distinguish GM130 molecules localized on the inactivated Golgi stacks from cytosolic GM130. We used a photoconvertible version of GM130 fused to Dendra2 (Gurskaya et al., 2006) and confirmed that it normally targets the Golgi apparatus (unpublished data). ManII-HRP cells expressing Dendra2-GM130 were cross-linked and observed by confocal microscopy (Figure 6, left panel). Dendra2-GM130 proteins localized on the inactivated Golgi membranes were then irreversibly identified: a region of interest was drawn around the cross-linked Golgi apparatus, and Dendra2-GM130 was then photoconverted to red only inside this region (Figure 6, middle panel). The same cells were imaged again 90 min later (Figure 6, right panel), and we observed that the photoconverted GM130 was also found on the re-formed Golgi-like structure, indicating that a fraction of GM130 was relocated from the inactivated Golgi to the new structure.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Brachet et al., 1999; Pond and Watts, 1999; Ang et al., 2004). However, it has so far relied on HRP-labeled probes captured from the extracellular medium and has thus been applicable only to organelles connected to the endocytic pathway. Here we show that this strategy can be extended to virtually any organelle if at least one specific marker is known that can be fused to HRP in its luminal domain. HRP must be expressed in the lumen of the compartment both to protect it from the reducing environment of the cytosol and to restrict the precipitation to the targeted organelle, which is essential to ensure specific inactivation. We observed that expression-based enzymatic organelle inactivation is endowed with the same level of specificity as previously described endocytic methods and represents a new tool to specifically perturb organelle function and dynamics.
One key question in cell biology is how are organelles inherited in mitosis and maintained in interphase. Can they form de novo or is "partitioning/replication" the rule? In the case of the Golgi complex, the question is particularly intriguing because organelle maintenance has to be considered in the context of the particular Golgi architecture and of the impressive flux of lipids and proteins transiting through the Golgi stacks in a bidirectional way. Two competing models have been proposed to explain intra-Golgi trafficking: according to the "static cisternae" model a stable Golgi structure receives, modifies, and sorts incoming cargos; in the "cisternal maturation" model that cargos are packed into Golgi cisternae that will progress in the direction of transport, stepwise maturing to acquire enzymes and markers of the next transportation stage (Beznoussenko and Mironov, 2002).
Most studies have addressed the question of Golgi re-formation in the context of cell division, which also led to competing models (Roth, 1999; Zaal et al., 1999; Jokitalo et al., 2001; Shorter and Warren, 2002; Barr, 2004). Interestingly, although interphase Golgi complex dynamics seem to be better described by the cisternal maturation model, which predicts efficient neo-formation of Golgi structures, the model most commonly used to describe its re-formation at mitosis exit is based on the fusion of pre-existing Golgi elements and does not rely on auto-organization. This may indicate that the mechanisms responsible for interphase Golgi apparatus growth and replication are different from the mechanisms responsible for its re-formation at the end of cell division.
We therefore asked the question of Golgi maintenance in interphase and analyzed how cells recover from Golgi inactivation and in particular whether a new Golgi apparatus can re-form de novo. Interestingly, new Golgi-like elements formed soon after Golgi cross-linking. We found that these structures are positive for newly synthesized secretory proteins, suggesting that they formed, at least partly, from the ER/intermediate compartment that migrated toward the MTOC area. However, these newly formed Golgi-like structures remain immature, both morphologically and functionally, which agrees with what was observed before in cytoplasts that lack the Golgi apparatus (Pelletier et al., 2000). First, these Golgi-like structures are incompetent for anterograde trafficking because VSV-G was not able to reach the plasma membrane and was not glycosylated by Golgi-specific enzymes. Second, they are not shaped like the mature organelle. No Golgi stacks were observed but, instead, enlarged vacuolar-like structures positive for GM130 were often detected. Third, although they are positive for most Golgi markers (see Table 1), important proteins like giantin or GS15 are missing.
The lack of giantin on these newly formed structures is reminiscent of its absence in dispersed mini-Golgi elements formed during nocodazole treatment (Nizak et al., 2003). This is particularly interesting because these elements were shown to be new Golgi apparatus formed from the ER after nocodazole addition (Cole et al., 1996; Storrie et al., 1998). However, when observed by EM, and in contrast with newly formed Golgi-like structures that we report here, they were shown to be composed of apposed cisternae, forming stacks very similar to normal Golgi, albeit less extended (Cole et al., 1996). This suggests that giantin is a marker of mature Golgi but is not necessary for stack apposition (see also Puthenveedu and Linstedt, 2001). Its absence from re-formed Golgi-like structures is thus unlikely to be responsible for the observed morphology defects. In contrast, because giantin has been implicated in COPI-dependent anterograde trafficking (Alvarez et al., 2001; Short et al., 2005), its lack may participate in the trafficking defects that we observed in these Golgi-like structures.
The other important marker missing on newly formed immature Golgi-like structures is GS15. Interestingly, its partners YKT6, GS18, and syntaxin5 (Xu et al., 2002) were detected on these structures indicating that although the t-SNAREs were able to reach the newly formed Golgi-like elements, the v-SNARE GS15 was not. This is surprising because Xu et al. (2002) found that GS15 is essential to ensure proper localization of the other members of the complex. However, it has been observed that these proteins can be localized on different structures. Volchuk et al. (2004) reported that, although syntaxin 5 and GS28 are homogeneously distributed across the Golgi apparatus, GS15 concentration increases toward the trans side. In addition, Tai et al. (2004) showed that, upon perturbation of recycling endosomes, GS15 can be trapped on endosomes, whereas syntaxin 5 and GS28 are still Golgi localized. Similarly we observed that after Golgi inactivation syntaxin 5 and GS28 can recycle to the newly formed Golgi-like structures, whereas GS15 cannot.
This would probably affect the function of what Volchuk et al. (2004) called the trans-SNAREpin, and it is thus temping to speculate that a key step necessary to re-form a mature and functional Golgi apparatus would involve this complex. A first possibility, as proposed by Volchuk et al. (2004) is that cis-SNAREpin (not containing GS15) and trans-SNAREpin are sequentially active during anterograde transport. Both steps would be necessary to ensure proper Golgi maturation and function, and because only cis-SNAREpins would be active after Golgi ablation, an immature Golgi-like structure would be formed. Another possibility takes into account that the trans-SNAREpin is involved in retrograde trafficking (Tai et al., 2004). Mature Golgi formation may thus rely on anterograde and retrograde countercurrent membrane flow (see Volchuk et al., 2004). To test these hypotheses, it will be interesting to inactivate GS15 and analyze by EM the structure of the dispersed Golgi apparatus obtained under these conditions (Xu et al., 2002). In addition, it will be important to test whether the re-formed Golgi-like structures can be reached by the GS15-dependent retrograde cargo Shiga toxin B subunit.
Altogether, our results suggest the following model for Golgi complex formation and maturation. The ER generates membranes, containing cargo en route to the Golgi-like VSV-G. These elements are recognized by microtubule minus-end–directed motors and move toward the center of the cell. Golgi peripheral proteins like GMAP210 or GM130 bind to these newly formed membranes, and at least a fraction of these proteins recycle from pre-existing Golgi elements. By themselves, these proteins cannot build functional and mature Golgi because re-forming Golgi generated after HRP-based inactivation are not transport-competent and are not normally shaped. Then, these immature Golgi elements acquire key factors (like GS15) that are presumably transported back to the newly formed Golgi structures from older Golgi cisternae, possibly in a membrane-based, ER-independent retrograde pathway. This will ensure efficient intra-Golgi transport (sequential and/or countercurrent flows), which is essential for the re-forming Golgi to acquire its normal apposed and flattened structure and to become transportation-competent. Whether morphology and activity are functionally linked is still unknown but will now be investigated using the Golgi inactivation system. Finally, additional factors reach the Golgi apparatus via the ER in an anterograde manner, upon neo-synthesis or after slow, ER-dependent recycling. This may be the case for giantin or newly synthesized proteins and possibly for TGN46 that appears on re-forming Golgi structures later than GM130.
In conclusion, the new organelle inactivation system that we describe here will be invaluable to study the function and dynamics of membrane-bound organelles. It allowed us to propose a two-phase integrative model for interphase Golgi complex formation where cargo-containing membranes emanating from the ER mature and acquire Golgi proteins in a stepwise manner, but will not organize as morphologically normal and active Golgi complexes until they receive material from, or fuse with, the pre-existing Golgi elements, hence suggesting that one needs a functional Golgi apparatus to make a new one.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Franck Perez (Franck.Perez{at}curie.fr)
Abbreviations used: BFA, brefeldin A; DAB, 3,3'-diaminobenzidine; EM, electron microscopy; EGFP, enhanced green fluorescent protein; EndoH, endoglycosidase H; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting; HRP, horseradish peroxidase; ManII, mannosidase II; TGN, trans-Golgi network; VSV-G, vesicular stomatitis virus G protein.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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