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First published online October 13, 2006; 10.1104/pp.106.090423 Plant Physiology 142:1442-1459 (2006) © 2006 American Society of Plant Biologists OPEN ACCESS ARTICLE
Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells1,[W],[OA]Department of Biology (Y.C.T., S.W.L., L.J.) and Molecular Biotechnology Program (Y.C.T., S.W.L., L.J.), The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; Department of Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, D–69120 Heidelberg, Germany (S.H.); and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom (P.D.)
Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5–10 µg mL–1) induced YFP-marked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50–100 µg mL–1) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose- and time-dependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.
All eukaryotic cells have secretory and endocytic pathways that are composed of several functionally distinct membrane-bound compartments, each with characteristic proteins that can be used as markers to define these compartments. Prevacuolar compartments (PVCs) or late endosomes are intermediate organelles where secretory and endocytic traffic leads to vacuole or lysosome merge (Bethke and Jones, 2000  ; Lemmon and Traub, 2000; Jiang and Rogers, 2003; Maxfield and McGraw, 2004; Lam et al., 2005). These prevacuolar or late endosomal compartments characteristically possess internal microvesicles and are thus called multivesicular bodies (MVBs) or endosomes. In animal cells, these microvesicles appear to originate in the early or recycling endosomes (Parton et al., 1992) and have a different protein composition to the limiting membrane (Griffiths et al., 1990; Kobayashi et al., 1998). The microvesicles and the soluble content of these MVBs are most likely delivered into the interior of the lysosomal compartment via direct fusion (Luzio et al., 2000; Katzmann et al., 2002).
In spite of the obviously important role of PVCs in mediating protein traffic to vacuoles in the secretory and endocytic pathways, the identification and characterization of plant PVCs, both functionally and morphologically, have been challenging due to the complexity of the plant vacuolar system, the existence of multiple transport pathways leading to distinct vacuoles, and the lack of good markers (antibody and reporter) to be used to define or identify PVCs (Jiang and Rogers, 1998
MVB-type organelles have been described some time ago in plant cells (Robinson et al., 2000
Similar to wortmannin, the fungal macrocyclic lactone brefeldin A (BFA) has been widely used in studying protein trafficking in the secretory and endocytic pathways of eukaryotic cells. In mammalian cells, BFA targets a subclass of Sec7-type guanine nucleotide exchange factors (GEFs) required for converting the GDP ribosylation factor 1 (Arf1p) to its GTP form (Jackson and Casanova, 2000
The Golgi apparatus is likely the initial site in response to BFA in both plant and mammalian cells (Sciaky et al., 1997
We previously demonstrated that VSR proteins can be used as markers to define plant PVCs (Li et al., 2002 In this study, we extended our understanding of BFA effects on PVCs/MVBs in plant cells. We used these two transgenic BY-2 cell lines expressing YFP/green fluorescent protein (GFP) reporters marking Golgi and PVC to test the hypothesis that BFA also affects other endomembrane organelles in addition to the ER and the Golgi apparatus. Here, we demonstrated that YFP-marked PVC and YFP-marked Golgi stacks showed different sensitivity in response to BFA treatment. BFA at low concentrations (5–10 µg mL–1) induced YFP-marked Golgi stacks, but not YFP-marked PVC, to form aggregates. However, BFA at higher concentrations (50 or 100 µg mL–1) induced both YFP-marked PVC and Golgi stacks to form typical enlarged structures in a dose- and time-dependent manner. Confocal immunofluorescence studies demonstrated that BFA-induced aggregates derived from YFP-marked PVCs colocalized with VSR proteins, but kept physically distinct from the Golgi-derived aggregates in BFA-treated cells. Further immunogold electron microscopy (immunoEM) and structural EM studies identified unique structures of Golgi- and PVC-derived aggregates. In addition, BFA also induced VSR-marked PVCs to form similar aggregates in root tip cells of pea, tobacco, mung bean (Vigna radiata), and Arabidopsis.
Development of the GFP BP-80 Transgenic Cell Line
We have previously demonstrated that VSR proteins are markers for defining PVCs in plant cells (Li et al., 2002
As a first step to compare reporter-marked Golgi to PVC directly in transgenic living tobacco BY-2 cells, we further developed a new transgenic tobacco BY-2 cell line expressing the GFP BP-80 fusion in which the GFP was used to replace the YFP in the previous YFP BP-80 construct (Tse et al., 2004
Golgi localization of the GONST1-YFP reporter and PVC localization of the GFP/YFP BP-80 reporter in transgenic BY-2 cells were further confirmed by immunoEM studies where GFP antibodies specifically labeled Golgi stacks (Fig. 1B, left image) or MVB (Fig. 1B, right image) in transgenic BY-2 cells expressing either Golgi or PVC reporters. Thus, similar to YFP BP-80, the GFP BP-80 reporter served as a tool for defining PVCs/MVBs in tobacco BY-2 cells. Therefore, from now on, both YFP BP-80 and GFP BP-80 cell lines were used as PVC markers in this study.
BFA has been a useful tool in studying protein trafficking in the secretory pathway because this drug induced Golgi stacks to form enlarged compartments and prevented protein traffic from the ER to the Golgi (Jiang and Rogers, 1998
We first performed a dose-response experiment in which day 3 transgenic cell lines expressing either the Golgi or PVC reporters were incubated with various concentrations of BFA (from 0–100 µg mL–1) for 1 h before the treated cells were collected and used in confocal imaging. Day 3 BY-2 cells have been used in our drug treatment studies because these cells are at their log phase (Matsuoka et al., 2004
BFA at 5 to10 µg mL–1 induced the GONST1-YFP-marked Golgi to form aggregates in transgenic BY-2 cells (Fig. 2A); this result is consistent with several previous studies showing BFA-induced Golgi aggregation in different cell types (Wee et al., 1998 ; Baldwin et al., 2001; Tse et al., 2004; Miao et al., 2006). However, several other studies showed that BFA at such concentrations also induced GFP-marked Golgi to form ER patterns in both BY-2 and tobacco leaf cells (Ritzenthaler et al., 2002; Saint-Jore et al., 2002). To find out the possible causes for such observed differences, we collected BY-2 cells at day 3 and day 6 for BFA treatment at 10 µg mL–1 for 1 h before confocal imaging. Day 3 and day 6 cells represent the log phase and the stationary phase, respectively (Matsuoka et al., 2004). As shown in Supplemental Figure S2, similar punctate patterns were observed in GONST1-YFP cells collected at either day 3 or day 6 (left images 1 and 6). At the end of 1-h BFA treatment, major aggregation patterns were observed from day 3 cells (left images 2 and 3) or day 2 cells (data not shown), but typical ER patterns were observed from BFA-treated day 6 cells (left images 5 and 6) or day 7 cells (data not shown), indicating that the physiological status of GONST1-YFP BY-2 cells may affect its responsive patterns to BFA treatment. However, when the same BFA treatments were performed using transgenic Man1-GFP BY-2 cells, ER patterns were observed from both BFA-treated day 3 (right images 2 and 3) and day 6 cells (right images 5 and 6). We do not know whether such observed differences are due to the difference between the trans-Golgi GONST1 and the cis-Golgi Man1 or to the different cell line used, but such differences shall not affect this study on the effects of BFA on PVCs/MVBs, where the GONST1-YFP cells were used as a control for XFP BP-80-marked PVC/MVBs in BY-2 cells (see "Discussion"). To study further the dynamics of the BFA-induced response of PVCs to high concentrations of BFA, a time-course study was carried out using these two transgenic cell lines. Day 3 individual transgenic cell lines were first incubated with BFA at 50 µg mL–1, a minimal concentration that induced PVCs to form aggregates (Fig. 2), followed by sample collection at indicated times after BFA treatment for confocal imaging. As shown in Figure 3A , in cells expressing the Golgi marker GONST1-YFP, enlarged BFA-induced aggregates were already detected 15 min after BFA treatment and these aggregated structures remained throughout the 2-h period of BFA treatment. In contrast, in cells expressing the PVC marker YFP BP-80, no BFA-induced YFP-marked aggregates were detected (Fig. 3, B and C) and the number of YFP-marked PVCs remained unchanged (Fig. 3D) during the first 30 min of BFA treatment, but similar aggregates were observed from 45 min after BFA treatment and these aggregates remained detectable thereafter for 2 h (Fig. 3, B and C). Thus, when comparing to Golgi stacks, a 30-min delay was observed for PVCs to form BFA-induced aggregates in response to BFA at 50 µg mL–1 under these conditions.
Recovery of PVCs from BFA Treatment
Low concentrations of BFA (typically between 5 and 10 µg mL–1) have been commonly used in studying protein trafficking and organelle dynamics in tobacco BY-2 cells (Nebenfuhr et al., 2002 To find out whether BFA at 50 or 100 µg mL–1 is toxic to BY-2 cells and causes permanent damage, we performed a recovery study. Day 3 transgenic cells expressing the Golgi marker GONST1-YFP and PVC marker GFP BP-80 were treated with BFA at 100 µg mL–1 for 1 h, followed by washing off the BFA with fresh medium and then sampling collections at indicated time points upon recovery for confocal imaging. As shown in Figure 4A , in cells expressing the Golgi marker GONST1-YFP, fluorescent signals in both aggregates and an ER pattern were detected 15 and 45 min after BFA washing, which was then followed by disappearance of aggregates, reappearance of punctate signals, and weakening of the ER pattern after 1 h, and a fully punctate pattern was eventually observed after 2 h (Fig. 4A). These results indicate that the Golgi pattern can be recovered fully upon BFA removal. Similar patterns of changes were also detected in cells expressing the PVC marker GFP BP-80 upon removal of BFA and, again, normal punctate signals were recovered after 2 h (Fig. 4B). Furthermore, the BFA-treated BY-2 cells during the recovery period still looked normal as judged by their appearance in the differential interference contrast (DIC) images. Similar results were obtained when cells were treated with BFA at 50 µg mL–1 for 1 h, followed by washing off BFA in the recovery study in these two cell lines (Supplemental Fig. S3). These results demonstrate that BFA at 50 or 100 µg mL–1 did not permanently damage the treated BY-2 cells and that the BFA-induced aggregates from either Golgi or PVC organelles can be fully recovered into typical punctate patterns upon removal of BFA. Therefore, both Golgi apparatus and PVCs in transgenic BY-2 cells demonstrated the ability of recovery even in high doses of BFA treatment at 50 or 100 µg mL–1.
BFA-Induced Compartments Derived from Golgi and PVC Remain Distinct, But Are Closely Associated
BFA at 5 to 10 µg mL–1 induced formation of aggregates that may represent ER-Golgi hybrid structures in BY-2 cells (Ritzenthaler et al., 2002 To find out whether BFA-induced aggregates derived from Golgi and PVC remain distinct or fusion upon BFA treatment, we performed the following immunofluorescent labeling study. Day 3 transgenic BY-2 cells expressing GONST1-YFP and YFP BP-80 reporters were treated with BFA at 50 µg mL–1 for 1 and 2 h, followed by fixation and labeled with VSR antibodies for subsequent confocal imaging. As shown in Figure 5 , the PVC/MVB marker VSR antibodies detected aggregates (red) in fixed transgenic cells expressing either the Golgi or PVC reporter (Fig. 5). Interestingly, in fixed transgenic BY-2 cells expressing the PVC marker YFP BP-80, the BFA-induced aggregates derived from the YFP-marked PVCs (green) colocalized (more than 90%) with aggregates detected by VSR antibodies (red; Fig. 5, image 1, arrowheads in merged image). In contrast, in fixed BY-2 cells expressing the Golgi marker GONST1-YFP, BFA-induced aggregates derived from the GONST1-YFP-marked Golgi (green) remained largely distinct (more than 90%) from the aggregates marked by the PVC marker VSR antibodies (red) at 1 or 2 h after BFA treatment (Fig. 5 images 2 and 3, arrowhead versus arrow in merged images). In addition, most of these aggregates derived from Golgi and PVC showed close association and tended to join together. These results demonstrate that BFA-induced aggregates derived from Golgi stacks are different from those derived from PVCs in BY-2 cells and that VSR proteins remain intact within the PVC-derived aggregates in BY-2 cells.
To further investigate the nature and relationship of BFA-induced aggregates derived from Golgi and PVC in response to BFA treatment, we then performed a time-course experiment using transgenic cells expressing the Golgi marker GONST1-YFP. These transgenic cells were first incubated with BFA at 50 µg mL–1, followed by sample collection and fixation at an indicated time point before the fixed cells were used in labeling with VSR antibodies for subsequent confocal imaging analysis. As shown in Figure 6 , BFA-induced aggregates marked by the Golgi marker GONST1-YFP (green) were already detected 10 min after BFA treatment, and these aggregates remained similar throughout the 2-h treatment period (Fig. 6, images 1–3; green). In contrast, the VSR-marked PVCs (red) remained unchanged during the first 10 min, whereas VSR-marked aggregates became visible and gradually increased in 2 h (Fig. 6, images 1–3; red). In addition, aggregates derived from Golgi and PVC remained distinct, but closely associated (Fig. 6, merged images 2 and 3). The differential response of Golgi and PVC to BFA treatment was consistent with results obtained from living cells (Figs. 2 and 3).
BFA Effects on the Golgi and PVC Are Pharmacologically Distinguishable
To find out whether the effect of BFA on the formation of PVC-derived aggregates is a primary effect on a PVC target or a secondary effect of transport from the Golgi to the PVC, we performed the following experiments using two phospholipase A2 inhibitors, ONO-RS-082 and bromoenol lactone (BEL), which have been used to inhibit the effect of BFA in mammalian cells (de Figueiredo et al., 1998
Morphology of BFA-Induced Golgi and PVC Aggregates Confocal immunofluorescent studies thus far demonstrate that BFA at high concentration (50–100 µg mL–1) induced both Golgi and PVC to form aggregates that are closely adjacent to each other. To further identify and study the morphological structures of these BFA-induced aggregates and their relationship, immunoEM studies were then carried out where GFP and VSR antibodies were used to detect Golgi stacks and PVCs, respectively, in transgenic GONST1-YFP BY-2 cells. Day 3 GONST1-YFP transgenic BY-2 cells were treated with BFA at 100 µg mL–1 for 1 h before they were fixed by glutaraldehyde and embedded in Lowicryl (HM20). Ultrathin sections were then prepared for immunoEM labeling. As shown in Figure 8 , normal Golgi apparatus (Fig. 8A) was specifically labeled by GFP antibodies in untreated control cells. In contrast, in BFA-treated cells, curved Golgi aggregates were labeled specifically by GFP antibodies (Fig. 8B), which may likely represent the BFA-induced aggregates derived from the Golgi apparatus.
Because no visible MVBs were detected in ultrathin sections prepared from Lowicryl (HM20) blocks, we therefore prepared samples using a high-pressure frozen/freeze substituted protocol for immunoEM studies (Tse et al., 2004 ). As shown in Figure 8, in the untreated control cells, multivesicular PVCs were clearly labeled by VSR antibodies and labeling was mainly on the peripheral regions (Fig. 8, C and D). Similarly, in BFA-treated cells, similar sizes (100–200 nm) of vesicles were also labeled specifically by VSR antibodies to their peripheral membranes (Fig. 8, E–H). Moreover, unlike untreated cells where PVC/MVBs were often identified individually, these VSR-labeled vesicles were often detected as groups that tended to pack together and thus might be derived from PVC aggregation or vesiculation (Fig. 8, E–I). However, these aggregated vesicles containing VSR proteins seem to lose their MVB appearance because no visible internal vesicles were detected from these VSR-labeled PVCs under these conditions (see, for example, Fig. 8, E and G).
To study further the details of these Golgi- or PVC-derived aggregates in response to BFA treatment, we then performed structural transmission electron microscopy (TEM) studies using conventional chemical fixation and subsequent embedding in Spurr's resin as previously described (Tse et al., 2004
BFA Induced VSR-Marked PVCs to Form Aggregates in Root-Tip Cells of Other Plants We have thus far demonstrated that BFA at high concentrations induced both Golgi and PVC to form aggregates in tobacco BY-2 cells and that aggregates derived from Golgi and PVC remained distinct, but closely associated. To find out whether such a BFA response is unique to BY-2 cells or can also be observed in other cell types, we performed additional BFA induction experiments using root-tip cells of various plants. Mature seeds of mung bean, pea, Arabidopsis, and transgenic tobacco expressing the Golgi marker GONST1-YFP were first germinated before the root tips were treated with BFA at 0, 10, and 100 µg mL–1 for 1 h, followed by fixation and immunolabeling with VSR antibodies. As shown in Figure 10 , typical punctate patterns were observed in tobacco root-tip cells expressing the Golgi marker GONST1-YFP, and these YFP-marked Golgi formed aggregates in the presence of BFA at either 10 or 100 µg mL–1 (Fig. 10, column 1), a result consistent with that observed in transgenic GONST1-YFP BY-2 cells. These results indicated that BFA at high concentrations induced the Golgi to form aggregates in tobacco root-tip cells. In contrast, BFA at low concentrations (0 or 10 µg mL–1) did not induce any changes in the VSR-marked PVCs, but BFA at high concentrations (100 µg mL–1) induced the VSR-marked PVCs to form aggregates in root-tip cells of mung bean, pea, and Arabidopsis (Fig. 10, columns 2–4). Moreover, PVC-derived aggregates in Arabidopsis root-tip cells (Fig. 9B) look similar to those in BY-2 cells (Fig. 9A). Therefore, BFA at high concentrations induced Golgi and PVC to form aggregates in root-tip cells of various plants in a way that is similar to that of BY-2 cells.
Transgenic BY-2 Cells Expressing the PVC Reporter Are Useful Tools for Studying the Dynamics of PVCs
Transgenic tobacco BY-2 cells expressing GFP-tagged organelle markers have been useful tools for studying the dynamics of various endomembrane organelles, including ER and Golgi apparatus (Satiat-Jeunemaitre et al., 1999
The Golgi apparatus provides a site for rapid action in both mammalian and plant cells where BFA induces Golgi to form an ER-Golgi hybrid or a BFA compartment. However, mammalian cells and plant cells have different sensitivity to BFA treatment. For example, BFA at 2.8 µg mL–1 was sufficient and specific to induce the formation of BFA compartments and block protein traffic from ER to Golgi in animal cells (Klausner et al., 1992
In addition to ER and Golgi apparatus, BFA may also act on endosomes in mammalian cells. For example, BFA treatment induced endosomes to become tubulated in mammalian cells (Hunziker et al., 1991
In this study, we extended our understating to BFA effects on the prevacuolar organelles using transgenic tobacco BY-2 cells expressing the PVC marker GFP BP-80 reporter. We demonstrated that PVCs/MVBs in BY-2 cells formed aggregates in response to BFA treatment at high concentrations (i.e. at 50–100 µg mL–1), but not at low concentrations (i.e. at 5–10 µg mL–1). Such high BFA concentrations were within reversible levels because the tested organelles recovered fully (based on the punctate fluorescent patterns) when the drug was washed off. Furthermore, the target of the BFA effect on Golgi and PVC is distinct because the phospholipase A2 inhibitor ONO-RS-082 specifically prevented PVCs, but not Golgi, from forming aggregates in the presence of BFA. Furthermore, the delayed observation of PVC-derived aggregates compared to Golgi-derived aggregates indicated that the Golgi apparatus might be more sensitive than PVC in response to BFA treatment, which might be due to the different ADP-ribosylation factor (ARF) GEFs (exchange factors for ARF GTPases) present in these two organelles (Geldner et al., 2003
In addition, PVC-derived aggregates as identified by VSR antibodies in confocal immunofluorescence remained distinct from Golgi-derived aggregates in the same BFA-treated cells. ImmunoEM and structural TEM studies further identified PVC-derived aggregates as clusters of VSR-labeled PVCs with similar size (about 100–200 nm in diameter), but lost the appearance of their internal vesicles. Such BFA effect on PVCs in BY-2 cells is different from the effect of wortmannin on PVCs in which wortmannin induced PVCs to form small vacuoles, but the enlarged vacuoles still contain visible, but reduced, internal vesicles (Tse et al., 2004 In addition, BFA-induced PVC-derived aggregates were not limited to BY-2 cells, but also observed in several other cell types, including root-tip cells of pea, tobacco, rice (Oryza sativa), and Arabidopsis. Therefore, it seems that the BFA effect on PVCs is a general response in plant cells. It is thus interesting and important to find out the possible physiological consequences of PVC changes in response to BFA treatment and the molecular mechanism of BFA action on plant PVCs.
In this study, we have shown that Golgi and PVC have different sensitivity to BFA, where BFA at low concentrations (5–10 µg mL–1) caused Golgi, but not PVC, to form aggregates, whereas high BFA concentrations (50–100 µg mL–1) induced both Golgi and PVC to form aggregates. Several recent studies also demonstrated that BFA induced endosomes to form aggregates in plant cells. The Arabidopsis GNOM protein (an exchange factor for ARF GTPases-ARF-GEFs), which plays an important role in mediating endosomal recycling, auxin transport, and plant growth in Arabidopsis, was found to locate to endosomes because this protein colocalized with the internalized FM4-64 endosomal marker 30 min after the uptake study (Geldner et al., 2003
In this study, day 3 or day 2 BY-2 cells after subcultures were used to study the effects of BFA on PVC/MVB in tobacco BY-2 cells mainly for two reasons. First, cells at these 2 d are at their log phase, thus representing physiologically healthy stages, whereas cells at day 6 or day 7 represent stationary stages (Matsuoka et al., 2004
When day 3 or day 2 GONST1-YFP BY-2 cells were treated with BFA at 10 µg mL–1 for 1 h, typical Golgi aggregates were observed (e.g. Fig. 2A). In addition, similar BFA-induced aggregates were also observed in root cells of transgenic tobacco expressing the GONST1-YFP (Fig. 10). Such BFA-induced Golgi aggregations have been documented by many previous studies in various cell types, including Arabidopsis root cells for the expressed mammalian sialytransferase (ST; Wee et al., 1998
In addition, the physiological status of cells may contribute to such variation. Indeed, when day 3 and day 6 GONST1-YFP BY-2 cells were subjected to BFA treatment at 10 µg mL–1 for 1 h, aggregation patterns and ER patterns were observed from these two stages of cells, respectively (Supplemental Fig. S2). However, when another BY-2 cell line expressing Man1-GFP was used, ER patterns were observed from both day 3 and day 6 cells after BFA treatment (Supplemental Fig. S2). However, we do not know whether such differences between GONST1-YFP and Man1-GFP BY-2 cell lines are due to the difference between the trans-Golgi localization of GONST1-YFP and the cis-Golgi localization of Man1-GFP, or due to their different sensitivity to BFA treatment, or, even though unlikely, due to the different localization of GONST1-YFP and Man1-GFP to different populations of the Golgi apparatus in BY-2 cells. Because BFA-induced aggregates for GONST1-YFP have been demonstrated by several studies in living onion epidermal cells (Baldwin et al., 2001
Several endomembrane organelles, including the ER and the Golgi, formed aggregates in response to BFA treatment, which also affects the transport of proteins from the ER to the Golgi in the secretory pathway. The development of transgenic tobacco BY-2 cell lines expressing GFP-marked PVCs and the identification of MVBs as PVCs have allowed us to study the dynamic response of PVCs to various drugs in living cells. For example, wortmannin treatment induced GFP-marked PVCs to form small vacuoles and thus provided a quick tool for defining multivesicular PVCs in tobacco BY-2 cells (Tse et al., 2004 ). In this study, we demonstrated that BFA at high concentrations (50 or 100 µg mL–1) induced both YFP-marked PVCs and Golgi to form typical enlarged aggregates, but the PVC-derived aggregates remained physically distinct from the Golgi-derived aggregates. In addition, the BFA-induced formation of aggregates derived from PVCs was specifically inhibited by a phospholipase A2 inhibitor ONO-RS-082, but this drug did not prevent the formation of BFA-induced Golgi-derived aggregates in BY-2 cells. The different changes of Golgi and PVC in response to wortmannin and BFA would provide quick tools to distinguish PVC from Golgi and identify PVC that will serve as a first step to study the molecular mechanism of the BFA effect on PVC-mediated protein traffic in the secretory and endocytic pathways of plant cells. Several questions can be addressed in future research. For example, what is the molecular target of BFA on PVC? Does BFA also affect PVC-mediated protein trafficking between Golgi and PVC or between PVC and plasma membrane? What are the molecular mechanisms of PVC-mediated protein trafficking in the plant secretory pathway? What is the physiological significance of BFA-induced PVC-derived aggregates in plant cells? Our current research is addressing some of these questions to further understand the roles of PVCs in plant cells.
General methods for construction of recombinant plasmids, characterization of cloned inserts, transformation of tobacco (Nicotiana tabacum) BY-2 cells, maintenance of transgenic tobacco BY-2 cells, and preparation and characterization of antibodies have been described previously (Jiang and Rogers, 1998 , 2001; Cao et al., 2000; Jiang et al., 2000; Tse et al., 2004). BY-2 cells were maintained by subculture twice a month on agar plates or every 7 d in liquid cultures at room temperature (21°C–22°C). Liquid cultures were kept in shakers at 125 rpm.
GFP with a signal peptide sequence from proaleurain (spGFP) was amplified by PCR and subcloned into the pYFP BP-80 construct via HindIII/EcoRI sites to replace the pYFP and resulted in pGFP BP-80 (Tse et al., 2004
For BFA treatment experiments, aliquots of BFA (stock at 2.5 mg mL–1 in dimethyl sulfoxide) solution were added to 2- and 3-d-old suspension cultures (log phase cultures) to give the proper final concentrations. BFA-treated cells were then removed from the cultures at indicated times for direct confocal imaging or fixed for confocal immunofluorescence and EM. For recovery experiments, BY-2 cells were treated with BFA at indicated concentrations, followed by centrifugations at low speed and washing three times with fresh Murashige and Skoog medium before samples were used for confocal imaging or fixation. On average, more than 100 cells were observed to obtain similar results for each experiment. All drug treatment experiments had been repeated at least two to three times with similar results to make sure that the drugs were functional and the cells used were at the same or similar physiological stages.
For drug treatment using OBO-RS-082 (BIOMOL) and BEL (Sigma), aliquots of the ONO-RS-082 and BEL (stock at 1 mM in dimethyl sulfoxide) solution were added to 2- to-3-d-old suspension cultures to give the proper final concentrations and incubated for 15 min, followed by addition of BFA at indicated final concentrations and incubated for 1 h. The treated cells were then collected and subjected to direct confocal imaging.
Production and characterization of VSR antibodies were described previously (Tse et al., 2004
Fixation and preparation of tobacco BY-2 cells, labeling, and analysis by epifluorescence and confocal immunofluorescence have been described previously (Jiang and Rogers, 1998
For samples embedded in LR White and Lowicryl (HM20) for immunoEM studies, cells were fixed in 1 mL of a primary fixative solution containing 0.25% (v/v) glutaraldehyde and 1.5% (v/w) paraformaldehyde in 50 mM phosphate buffer, pH 7.4, for 15 min at room temperature and then transferred to 4°C for an additional 16 h. After washing with phosphate buffer at room temperature, cells were dehydrated in an ethanol series and then embedded in LR White and Lowicryl (HM20) resin. For samples embedded in Spurr's resin for structural TEM studies, cells were fixed in 1 mL of a primary fixative containing 2% (v/v) glutaraldehyde and 0.1 mL of saturated picric acid in 25 mM CaCo buffer, pH 7.2, for 15 min at room temperature and then transferred to 4°C for an additional 16 h. After washing with 25 mM CaCo buffer, pH 7.2, cells were further subjected to a secondary fixative solution containing 2% (w/v) osmium tetroxide and 0.5% (w/v) potassium ferrocyanide in 25 mM CaCo buffer, pH 7.2, for 2 h at room temperature. The cells were then washed with 25 mM CaCo buffer, pH 7.2, followed by contrasting at 2% aqueous uranyl acetate for 2 h at room temperature. After washing twice in water, cells were dehydrated in an acetone series and finally embedded in Spurr's resin.
For sample preparation using high-pressure frozen/freeze substituted, procedures were performed essentially as described previously (Tse et al., 2004
The following materials are available in the online version of this article.
We are grateful to Prof. David G. Robinson (University of Heidelberg) and John C. Rogers (National Science Foundation) for their continuous support of our studies. A portion of this work has been presented in abstract form for a poster for the American Society of Plant Biologists Annual Meeting 2005 (http://abstracts.aspb.org/pb2005/public/P51/7276.html). Received September 27, 2006; accepted October 9, 2006; published October 13, 2006.
1 This work was supported by the Research Grants Council of Hong Kong (grant nos. CUHK4156/01M, CUHK4260/02M, CUHK4307/03M, and CUHK4580/05M), the National Science Foundation of China (grant no. 30529001), and the Chinese University of Hong Kong Scheme C (to L.J.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Liwen Jiang (ljiang{at}cuhk.edu.hk).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.090423 * Corresponding author; e-mail ljiang{at}cuhk.edu.hk; fax 852–2603–5646.
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-COPI antibodies in confocal immunofluorescence (Ritzenthaler et al., 2002
C:mRFP (Kong et al., 2006
