ARTICLE |
Correspondence to: Azeddine Driouich, CNRS UMR 6037-IFRMP 23, Université de Rouen, UFR des Sciences, Centre Commun de Microscopie Electronique, 76821 Mont St Aignan Cedex, France. E-mail: Azeddine.Driouich@univ-rouen.fr
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells. (J Histochem Cytochem 51:931940, 2003)
Key Words: Golgi, green fluorescent protein, glycosyltransferases, immunogold, microscopy, plant
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN PLANT CELLS, the secretory system (endoplasmic reticulum, Golgi apparatus, transport vesicles) is involved in processing, sorting, and transport of glycoproteins and non-cellulose matrix polysaccharides of the cell wall (
A central issue for immunolocalization at the EM level is to preserve the epitope of interest while minimizing the loss of ultrastructural details. There are several strategies for the preparation of biological samples destined for immunogold labeling of proteins. These include (a) chemical fixation with aldehyde followed by progressive dehydration and embedding under low-temperature conditions (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Growth Conditions
Cells were harvested by low-speed centrifugation (for 2 min) 35 days after subculturing and immediately used for experiments.
Construction of the XylT36::GFP and Transformation of Plant Cells
The fusion protein consisting of the first 36 amino acids of AtXylT (ß1-2 xylosyltransferase from Arabidopsis thaliana) fused to the GFP (XylT36::GFP) was constructed as described in
Production of Antibodies Directed Against GFP
The specific anti-GFP polyclonal antibodies were prepared in rabbits using commercial GFP (Q-biogen; Illkirch, France). The procedure of antibody preperation was as described in
Protein Extraction, SDS-PAGE, and Western blotting Analysis
Cell extracts were prepared as described in
Fluorescence Microscopy
Five-day-old tobacco BY-2 cells expressing the fusion protein XylT36::GFP were observed using the standard fluorescein isothiocynate filter set. Image acquisitions were done with a Leica DMRB microscope coupled to an ORCA-ER CCD camera (C4742-95-12ER, Hamamatsu).
Sample Preparation for Electron Microscopy
Transgenic BY-2 cells expressing GFP-tagged Golgi glycosyltransferase were fixed in the dark for 30 min, 60 min, or 120 min at 4C in a solution consisting of 1% glutaraldehyde and 1% osmium tetroxide in 0.1 M Na cacodylate buffer, pH 7.2. This mixture was prepared and used immediately. After washing in distilled H2O, the samples were gradually dehydrated in 10%, 20%, and 40% aqueous ethanol (10 min in each bath), then in 60% and 80% (20 min in each bath), and finally in anhydrous ethanol for 30 min. After dehydration the samples were embedded in London Resin White (LRW) or Spurr resin. For the embedding in Spurr, cells were incubated twice in anhydrous acetone (5 min each), then infiltrated overnight at 4C in a mixture of Spurr and acetone (1/1) (v/v). Finally, the cells were bathed twice in pure and fresh resin (1 hr each). Polymerization was carried out at 60C for 24 hr. For the embedding in LRW, fixed and dehydrated cells were infiltrated at 4C in a mixture of LRW and ethanol, (1/2) (v/v) for 1.5 hr and (2/1) (v/v) for 1 hr. The cells were then incubated twice in pure resin overnight followed by 4 hr at 4C. Polymerization was carried out at 4C under UV light for 48 hr.
Electron Microscopy and Immunogold Labeling
Ultrastructural Observations.
Ultrathin sections (8090 nm) from Spurr and LRW-embedded samples were collected on formvar-coated copper grids and post-stained in 2% aqueous uranyl acetate and Reynold's lead citrate. Sections were examined at 80kV in a Philips electron microscope (Technai 12) at CCME (Rouen University; Rouen, France).
Detection of Complex N-glycans and Cell Wall Polysaccharides.
The antibodies used were the rat monoclonal antibody JIM7 (
Immunolocalization of GFP. The anti-GFP antibody used in this study is a rabbit polyclonal serum produced in our laboratory. Formvar-coated 300-mesh nickel grids carrying ultrathin sections (90 nm) of LRW-embedded transgenic tobacco BY-2 were incubated in 0.5 M Na-metaperiodate (NaIO4) for 30 min to remove excess fixatives. After washing in distilled H2O (twice for 10 min), grids were transferred to a 0.1 N HCl solution for 10 min to remove excess osmium tetroxide. Grids were then washed briefly and first blocked in 0.1 M glycine in PBS (0.01 M Na-phosphate, pH 7.2, and 0.15 M NaCl) containing 0.1% (v/v) Triton X-100 for 15 min to inactivate residual aldehydes. After washing in PBS containing 0.1% (v/v) Triton X-100 for 5 min, sections were blocked with the second blocking buffer solution consisting of normal goat serum (British Biocell; Cardiff, UK) 1:30 in PBS containing 0.1% (w/v) BSA and 0.2% (v/v) Tween-20 for 45 min. Grids were then incubated in the primary anti-GFP antibodies diluted 1:100 in the second blocking buffer at 4C overnight. A control was performed under the same conditions with the rabbit preimmune serum diluted 1:100 in the first blocking buffer solution. The grids were then washed once in PBS containing 0.1% (v/v) Triton X-100 for 5 min, then twice in TBS (0.05 M Tris-HCl, pH 8.4, and 0.15 M NaCl) containing 0.1% (v/v) Triton X-100 for 15 min each, and blocked for 45 min with the third blocking buffer solution consisting of normal goat serum (British Biocell) 1:30 in TBS containing 1% (w/v) BSA, 0.1% (v/v) Triton X-100, and 0.2% (v/v) Tween-20. The sections were then transferred to a droplet of goat anti-rabbit IgG conjugated to 10-nm gold particles (British Biocell) diluted 1:25 in the third blocking buffer solution for 2 hr at room temperature. After three washes in PBS containing 0.1% (v/v) Triton X-100 for 20 min and two washes in distilled H2O for 20 min, grids were finally post-stained and examined as described above.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ultrastructural Features of Tobacco BY-2 Cells
We first examined the ultrastructural features of tobacco BY-2 cells co-fixed with a glutaraldehyde/osmium mixture and embedded in Spurr resin (Fig 1). After 30 min of fixation, most if not all cell compartments were well preserved. The endomembranes, including nuclear envelope, the plasma membrane, tonoplast, Golgi stacks, and the endoplasmic reticulum (ER) were consistently discernable (Fig 1A and Fig 1B). A slight swelling of the ER, which is a common phenomenon of fixation, was observed at different times of fixation (Fig 1A 1C, and 1E). In the context of this article, it is worth noting that the organization of Golgi stacks was typical of plant suspension-cultured cells (
|
We also examined the ultrastructure of the same cells embedded in LRW resin, which is more amenable to immunogold labeling. In LRW-embedded cells, although some cytoplasmic extraction was observed, Golgi stacks and ER were quite well preserved and clearly distinguishable. As illustrated in Fig 2, even though the quality of structural preservation did not match that of the Spurr-embedded cells (Fig 1 and Fig 2B), the cis-to-trans polarity of Golgi stacks as well as subtypes of Golgi cisternae were identifiable. In most cases, the best preservation of these Golgi structures in LRW-embedded samples was obtained at a fixation time of 120 min (Fig 2A) vs 60 or 30 min of fixation.
|
Immunolocalization of Complex N-glycans and Cell Wall Polysaccharides
Before the immunolocalization of GFP, we checked whether the co-fixation protocol allowed classical immunogold labeling of plant cell wall polysaccharides and N-glycans both in the cell wall and Golgi stacks of tobacco BY-2 cells. As shown in Fig 3A and Fig 3B, specific and heavy immunogold labeling was found over Golgi cisternae and the cell wall using antibodies directed either against xyloglucan, the major hemicellulose polysaccharide in primary walls of dicots (
|
Immunolocalization of Golgi-targeted GFP
Specificity of the Antibodies.
Before investigating the immunogold localization of GFP on transformed tobacco BY-2 cells, we checked for the specificity of anti-GFP antibodies that we had produced in our laboratory, using immunoblotting technique. A total extract of intracellular proteins from non-transformed and transformed suspension-cultured BY-2 tobacco cells and a purified GFP, which was used for immunization, were probed on a Western blot with anti-GFP antibodies. As shown in Fig 4, the purified GFP (29 kD), was also detected with the anti-GFP antibodies (Fig 4, Lane 1). Although no signal was obtained with the protein extract of wild-type tobacco cells (Fig 4, Lane 2), anti-GFP antibodies detected a major polypeptide in the intracellular fraction of transformed cells (Fig 4, Lane 3). This polypeptide, of
32 kD, corresponds to the fusion protein AtXylT36::GFP. No immunostaining was observed with the preimmune control serum (not shown). These data demonstrate the specificity of anti-GFP antibodies.
|
Immunogold Localization of GFP. To investigate the immunogold localization of GFP, sections of transformed LRW-embedded BY-2 cells were immunolabeled with antibodies directed against GFP. We first performed and quantified the immunogold labeling over Golgi cisternae before and after a treatment with NaIO4 and HCl. The data are presented as a histogram in Fig 5. In untreated samples, binding of anti-GFP antibodies to the Golgi cisternae was weak, with most of the stacks carrying about three gold particles. In contrast, after NaIO4/HCl treatment, a much heavier labeling, four- to sevenfold higher than in untreated sections, was seen over the Golgi stacks. This demonstrates that the treatment was necessary to achieve significant and specific labeling of GFP within Golgi stacks in LRW-embedded cells. In addition, the density of GFP labeling seemed to be affected by the duration of fixation (Fig 5), with the highest density obtained with 60 min-fixed samples. When Spurr-embedded samples were used, GFP immunogold labeling was difficult to achieve and no significant labeling was obtained even after treatment with NaIO4 and HCl (data not shown).
|
Fig 6B illustrates the typical immunogold labeling pattern obtained with anti-GFP antibodies on LRW-embedded transformed BY-2 cells. Labeling was mostly found over the medial cisternae, with less over the cis and trans cisternae. The ER, tonoplast, and the vacuole were never labeled (Fig 6B). Scattered and very weak labeling was found over the plasma membrane and the cell wall with both anti-GFP and preimmune sera. Finally, no significant labeling was found in control experiments performed either using preimmune serum (Fig 5) or with the omission of the primary antibodies (not shown).
|
Together, the immunogold labeling results confirm that the punctate pattern of GFP fluorescence in BY-2 cells shown in Fig 6A corresponds indeed to dispersed individual Golgi stacks and that the fusion protein was targeted to the Golgi apparatus. In addition, quantitative analysis of the binding pattern of GFP demonstrates that the ß1,2 xylosyltranferase involved in N-glycan processing is mostly located in medial cisternae of Golgi stacks in tobacco BY-2 cells (see also
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fixation Protocols and Ultrasructural Preservation
Many chemical fixation methods are currently used to prepare specimens for immunogold labeling of proteins at the EM level (
Immunogold Labeling of Golgi-targeted GFP
We demonstrate that antibodies recognizing both carbohydrate epitopes and GFP bind to Golgi stacks of tobacco BY-2 cells prepared by the co-fixation method. First, immunogold labeling of cell matrix polysaccharides and complex N-glycans was possible and the quality of Golgi labeling with anti-ß1-2 xylose, anti-xyloglucan, and anti-pectin antibodies, did not differ from those previously observed in many plant cells either chemically or cryofixed (
The glycosyltransferase fused to GFP is a ß1,2 xylosyltransferase from Arabidopsis thaliana, which is a type II membrane protein located, along with all other glycosyltransferases involved in N-glycan processing and complex polysaccharide synthesis, in Golgi membranes (
In summary, we developed a simple co-fixation method of tobacco BY-2 suspension-cultured cells that provides a good preservation of cell ultrastructure while maintaining antigenicity of complex polysaccharides, N-glycans, and proteins of the Golgi membranes. We also show that the co-fixation method allows a specific localization of Golgi-targeted GFP using immunogold EM and NaIO4/HCl etching treatment. Although the HPF/FS remains the best technique for such purposes, the chemical co-fixation and immunocytochemical methods described here can be easily used to study GFP-tagged plant proteins of the endomembrane system in laboratories for which specialized HPF equipment is not available.
![]() |
Acknowledgments |
---|
Supported by the CNRS and the University of Rouen.
We are grateful to Dr Anja Geitman (University of Laval), to Dr Thomas Giddings (University of Colorado), and to John Moore (University of Cape Town) for helpful comments and critical reading of the manuscript. Thanks are also due to Laurence Chevalier and Jean Herbet for technical assistance and help at the Centre Commun de Microscopie Electronique (Rouen University).
Received for publication January 16, 2003; accepted March 5, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Batoko H, Zheng HQ, Hawes C, Moore I (2000) A rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants. Plant Cell 12:2201-2217
Boevink P, Oparka K, Santa Cruz S, Martin B, Betteridge A, Hawes C (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441-447[Medline]
Brandizzi F, Frangne N, MarcMartin S, Hawes C, Neuhaus JM, Paris N (2002a) The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain. Plant Cell 14:1077-1092
Brandizzi F, Fricker M, Hawes C (2002b) A greener world: the revolution in plant biomaging. Mol Cell Biol 3:520-530
Brorson SH (1998) Antigen detection on resin sections and methods for improving the immunogold labeling by manipulating the resin. Histol Histopathol 13:275-281[Medline]
Carlemalm E, Garavito RM, Villiger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microsc 126:123-143
Craig S, Goodchild DJ (1984) Periodate-acid treatment of sections permits on-grids immunogold localization of pea seed vicilin in ER and Golgi. Protoplasma 122:35-44
Criqui MC, Weingartner M, Capron A, Parmentier Y, Shen WH, HeberleBors E, Bögre L et al. (2001) Sub-cellular localization of GFP-tagged tobacco mitotic cyclins during the cycle and after spindle checkpoint activation. Plant J 28:569-581[Medline]
Driouich A, Faye L, Staehelin LA (1993) The plant Golgi apparatus: a factory for complex polysaccharides and glycoproteins. Trends Biol Sci 18:210-214
Driouich A, Staehelin LA (1997) 7-dehydrobrefeldin A, a natural-occuring BFA derivative, inhibits secretion and causes a cis-to-trans breakdown of Golgi stacks in plants cells. Plant Physiol 113:487-492
Faye L, Gomord V, FitchetteLainé AC, Chrispeels MJ (1993) Affinity purification of antibodies specific for Asn-linked glycans containing 1,3 fucose or ß1,2 xylose.. Ana Biochem 209:140-180
Fitchette AC, CabanesMacheteau M, Marvin L, Martin B, SatiatJeunemaitre B, Gomord V, Crooks K et al. (1999) Biosynthesis and immunolocalization of lewis a-containing N-glycans in the plant cell. Plant Physiol 121:333-343
Frigerio L, Vine ND, Pedrazzini E, Hein MB, Wang F, Ma JKC, Vitale A (2000) Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants. Plant Physiol 123:1483-1493
Geelen DNV, Inzé DG (2001) A bright future for bright yellow-2 cell culture. Plant Physiol 127:1375-1379
Gomord V, Denmat LA, FitchetteLainé AC, SatiatJeunemaitre B, Hawes C, Faye L (1997) The C-terminal HDEL sequence is sufficient for retention of secretory in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER. Plant J 11:313-325[Medline]
Gomord V, Fitchette AC, Denmat LA, Michaud D, Faye L (1998) Production of foreign proteins in tobacco cell suspension culture. In Cunningham C, Porter AJR, eds. Methods in Molecular Biotechnology. Vol 3. Totowa, NJ, Humana Press, 155-164
Granger CL, Cyr RJ (2000) Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD. Planta 210:502-509[Medline]
Hayat MA (1970) Polymerization. In Principles and Techniques of Electron Microscopy. New York, Van Nostrand Rheinhold
Hayat MA (2000) Chemical fixation. In Principles and Techniques of Electron Microscopy. Biological Applications. 4th ed. Cambridge, Cambridge University Press
Höfgen R, Willmitzer L (1998) Storage of competent cells for Agrobacterium transformation. Nucleic Acid Res 16:9877
Keegstra K, Raikhel N (2001) Plant glycosyltransferases. Cur Opin Plant Biol 4:219-224[Medline]
Knox JP, Linstead PJ, King J, Cooper C, Roberts K (1990) Pectins esterification is spatially regulated both within cell walls and between developing tissues of root apices. Planta 181:512-521
Kost B, Spielhofer P, Chua NH (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J 16:393-401[Medline]
Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680-685[Medline]
Lainé AC, Gomord V, Faye L (1991) Xylose-specific antibodies as markers of subcompartmentation of terminal glycosylation in the Golgi apparatus of sycamore cells. FEBS Lett 295:179-184[Medline]
Lynch M, Staehelin LA (1992) Domain-specific and cell-specific localization of two types of cell wall matrix polysaccharides in the clover root tip. J Cell Biol 118:467-479[Abstract]
Moore PJ, Darvill AG, Albersheim P, Staehelin LA (1986) Immunogold localization of xyloglucan and rhamnogalacturonan 1 in the cell wall of suspension-cultured sycamore cells. Plant Physiol 82:787-794
Moore PJ, Swords KMM, Lynch MA, Staehelin LA (1991) Spatial organization of the assembly pathway of glycoproteins and complex polysaccharides in the Golgi apparatus of plants. J Cell Biol 112:589-602[Abstract]
Nebenführ A, Gallagher LA, Dunahay TG, Frohlick JA, Mazurkiewicz AM, Meehl JB, Staehelin LA (1999) Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol 121:1127-1141
Nikus J, Daniel G, Jonsson L (2001) Subcellular localization of beta-glucosidase in rye, maize and wheat seedlings. Physiol Plant 111:466-472[Medline]
Pagny S, Bouissonié F, Sarkar M, FolletGueye ML, Driouich A, Schachter H, Faye L et al. (2003) Structural requirements for Arabidopsis ß1,2xylosystransferase activity and targeting to the Golgi. Plant J 33:189-203[Medline]
Paupard MC, Miller A, Grant B, Hirsh D, Hall DH (2001) Immuno-EM localization of GFP-tagged Yolk proeins in C. elegans using microwave fixation. J Histochem Cytochem 49:949-956
Quintana C (1994) Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies. Micron 25:63-99[Medline]
Ritzenthaler C, Nebenführ A, Movafeghi A, StussiGaraud C, Behnia L, Staehelin LA, Robinson DG (2002) Reevaluation of the effects of brefeldin A on plant cells using tobacco Bright Yellow 2 cells expressing Golgi-tagged green fluorescent protein and COPI antisera. Plant Cell 14:237-261
Robinson DL, Kahn ML, Vance CP (1994) Cellular localization of nodule-enhanced aspartate aminotransferase in Medicago sativa L. Planta 192:202-210
Sakurai MH, Kiyohara H, Tsumuraya Y, Hashimoto Y, Yamada H (1998) Characterization of antigenic epitopes in anti-ulcer polysaccharides from Bupleurum falcatum L. using several carbohydrases. Carbohydr Res 311:219-229[Medline]
Staehelin LA, Giddings TH, Jr, Kiss JZ, Sack FD (1990) Macromolecular differentiation of Golgi stacks in root tip of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples. Protoplasma 157:75-91[Medline]
Tokuyasu KT (1973) A technique for ultracryomicrotomy of cells suspensions and tissues. J Cell Biol 57:551-565
Vicré M, Jauneau A, Knox JP, Driouich A (1998) Immunolocalization of ß1,4 and ß1,6-D-galactan epitopes in the cell wall and the Golgi stacks of developing flax root tissues. Protoplasma 203:26-34
Wee EGT, Sherrier DJ, Prime TA, Dupree P (1998) Targeting of active sialyltransferase to the plant Golgi apparatus. Plant Cell 10:1759-1768
Winicur ZM, Zhang GF, Staehelin LA (1998) Auxin deprivation induces synchronous Golgi differentiation in suspension-cultured tobacco BY-2 cells. Plant Physiol 117:501-513
Zhang GF, Driouich A, Staehelin LA (1993) Effect of monensin on plant Golgi: re-examination of the monensin-induced changes in cisternal architecture and functional activities of the Golgi apparatus of sycamore suspension-cultured cells. J Cell Sci 104:819-831
Zhang GF, Staehelin LA (1992) Functional compartmentalization of the Golgi apparatus of plant cells. An immunochemical analysis of high pressure frozen and freeze substitued sycamore maple suspension-cultured cells. Plant Physiol 99:1070-1083
Zuo J, Niu QW, Wu Y, Kost B, Chua NH (2000) KORRIGAN, an Arabidopsis endo-1,4-ß-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137-1152