- Email: [email protected]
Visualization of microtubules in walled plant cells by immunogold silver-enhancement
Cell Biology
International
Reports,
Vol. 14, No. 12, December
1990
1109
VISIJAI,IZATION OF MICROTURUIXS IN WAI,IXD PI,ANT
0309-l
651/90/l
21109-l
O/SO3.00/0
0 1990 Academic Press Ltd
1110
Cell Biology
international
Reports,
Vol. 14, No. 12. December
1990
enhancement procedure (IGSS) based on the techniques developed by Dansher and Niirgaard (1983) and Holgate et al. (1983). In order to visualise and to discuss the cell wall modifications induced by the enzymatic permeabilisation, cell wall ultrastructure has been checked by electron microscopy after polysaccharide staining. MATERIAI,
AND METHODS:
Plant material. Seedsof mung bean (Vigna rdiatu) were immersed for 4 h in distilled water, and allowed to germinate on moistened vermiculite in plastic boxes at 26 C in darkness. When the shoots were 3 days old, strips from the growing part of the hypocotyl (just below the hook) were pealed as described below. Preparations of tissues for whole cell immunocytochemistry. Strips of epidermal cells were pealed from the hypocotyls under fixative for 45 min in freshly prepared 3% paraformaldehyde in microtubule-stabilising buffer (MSB), pH 6.8 (MSB = 100 mM PIPES, 10 mM EGTA, 5 mM MgS04). Strips were then immersed for 15 min in 1% NaBH4 in MSB in order to saturate free aldehyde groups and washed for 45 min in MSB. A partial cell wall digestion was performed by an 8-10 min treatment with 2% cellulase (Sigma) and 1% macerozyme R-10 (Yakult biochemicals co.) in MSB, followed by a 30 min wash in MSB. Preservation of cellular shapewas checked in a few specimens after staining with Calcofluor white. A 10 min immersion in 1% Nonidet NP40 (Sigma) in MSB was performed to permeabilise the plasma membrane. Strips were washed again in MSB, teased flat onto coverslips with needles and allow to air dry overnight. A progressive and perfect air drying was generally enough to achieve satisfactory adhesion of the strips onto the coverslips. For immunofluorescence observations, adhesion was improved by prior treatment of the coverslips with a 5 mg/ml poly-L-lysine solution (MW > 300 000, Sigma) (coverslips were left in contact with the poly-L-lysine solution for 3 h and then washed in distilled water). The slides were used either wet or dry. Immunocytochemistry. The immunocytochemical reaction was adapted from the protocols of Lloyd and Wells (1985) and Simmonds et al. (1985) as follows: in order to reduce background staining preparations were first incubated for 15 min in normal serum of the productive wies of the second antibody. The primary antibody (monoclonal antibodies to tubulin) was applied for 3-12 h at 26 C, followed by 1 h washing in 1% fish gelatin (Sigma) in MSB. Preparations were then incubated for 1 h at 26 C in darkness with a second antibody conjugated to a fluorescent probe (FITC or Texas red) or to 5 nm gold particles depending on the method of visualisation. Several ways of microtubule detection were used in order to spot differences in microtubule organisation: Immunofluorescence: a) Rat monoclonal antibodies to a tubulin at 1:15 dilution (Sera Lab distributed by Biosys, France), revealed by FITC rabbit anti-rat antibodies at 1:4OO(Biosys). b) Mouse monoclonal antibodies to @tubulin at 1:400 (Amersham) revealed either by FITC-conjugated sheep anti mouse antibodies at 1: 150 dilution or by Texas redconjugated sheepanti mouse antibodies at 1:80 dilution (Amersham).
Cell Biology
International
Reports,
Vol. 14, No. 12. December
1990
1111
Immunogold staining: Mouse monoclonal antibodies to B tubulin at 1:400 (Amersham) and secondly layer of 5 nm gold conjugated goat anti-mouse serum at 1:15 dilution (Amersham). I,ight Microscopy. Slides prepared for fluorescence microscopy were washed in I % fish gelatin in MSB for 1 h. and mounted in Citifluor (City University. London). They were observed with a Zeiss Axiophot epifluorescnece microscope. with specific FITC filter set or rhodamine filter set for Texas red. Slides prepared for IGSS were washed with filtered distilled water. The gold label was silver enhanced according to the technique of Dansher er al. (1983) with the Janssenintense 2 kit. Preparations were then mounted in Eukitt. Micrographs were taken on Kodak Thlax 400 or Ilford HP5 films. Electron microscopy and cell wall staining. The techniques used were adapted from Roland and Vian ( 1979). Strips of epidermal cells were pealed from the upper part of the hypocotyl (exponential growing area area. below the hook). Specimens were fixed in 4 % glutaraldehyde in 0.1 M sodium cacodylate buffer. pH 6.8, and washed in the samebuffer. A partial cell wall digestion was performed by an 8- 10 min treatment in enzymatic mixture as for light microscopv described above. followed by a 30 min wash in buffer. Specimens were dehydrated in a graded water/alcohol series and embedded in Spurr resin. In order to visualize the cell wall texture. ultrathin sections were treated with periodic acid/thiosemicarbazide/silver proteinate. (P.A.T.Ag). the cytochemical test for polysaccharides (Thiery 1967). Preparations were examined with a JEOL 1200EXIl transmission electron microscope.
RESULTS
AND DISCUSSION:
Comparison of IMF and IGSS techniques. As IMF techniques are currently used to detect microtubules in permeabilised cells they have been used here as a control for the IGSS techniques. Figure 1 shows immunofluorescence staining, at the tissue level, of microtubules in hypocotyl epidermis. Although the staining is rarely homogeneous through the whole tissue, the microtubular network is well detected (cf also Fig. 1 in Roberts CTal., 1985, and Fig. 5 a-c in Satiat-Jeunemaitre, 1989). Cortical microtubules appear organ&d in parallel arrays around the cells, in transverse, oblique or longitudinal directions according the cells observed. In Figure 2, a similar microtubular network is observed by immunogold staining after silver enhancement (see also Figs 5 d-f in SatiatJeunemaitre, 1989). It appears from these observations that the non-specific background staining is less of a problem with the immunogold staining technique than with immunofluorescence. Closer observations on microtubular arangements were performed on isolated cells (Figs. 3 & 4). Whatever the immunostaining combination used, the pattern of microtubule distribution was similar. There are no differences between staining with complexes directed against alpha- or beta-tubulin (cf Figs. 1 & 3 or 3 & 4). Again, IGSS and IMF techniques give similar information on the microtubular cytoskeleton:
1112
Cell Biology
International
Reports,
Vol. 14, No. 12, December
I990
Cell Biology
International
Reports,
Vol. 74, No. 12, December
1990
1113
1114
Cell Biology International
Reports, Vol. 14, No, 12, December
7990
Compurison of luhellin~ hg IMF or IGSS on isoluted walled cells. FiG. 3. (unti-atuhulin, FITCH. A honnqeneous and re~nlar urrun~etnent of tnicrotuhules uround the cell is revealed. Diflerent.focal planes (A und Bi .sholr* thur tnicrotuhules are arrunxed in purullel and continotts trun.s\‘er.se urruy. Note that both anti-hew und -ulphu tuhulin give sitnilar stainin~s puttems. X 650. Firr.4. Helical arrangewent of microtubules around a cell is revealed by \wiutions in .focal plunes. The continuity of rnicrotubule arruys,Potn the superior plune (A) to the inferior plune (B) of the cell.follows an helix. X 720. _____---________________________________-----------------------------------------------------~-------~-
different focal planes show that the cortical microtubules roll around the cells (Figs. 3 & 4) in parallel arrays. Where the microtubule lay in more oblique directions (Fig. 4). variations in the focal plane reveal a helical arrangement of the cortical microtubules. Since Lloyd’s first descriptions on microtubules in walled plant cells. many reports have shown that microtubules occur in stable helices in many growing cells ( Traas et ul. 1985; Roberts et al. 1985: Smith-Huerta & Jemstedt 1989: see Iwata & Hogetsu 1988, for more references).As they give similar results, either the IMF or IGSS techniques can be used in microtubule studies according the tissues studied or the aim of the observation. At the LM level, in walled ceils, IMF allows double staining with the use of different fluorochromes. but has two major disadvantages: firstly fluorochromes are sensitive to light and fade quite rapidly during observation. and secondly they must be avoided in plant tissues showing any autofluorescence. IGSS techniques obviate these two problems. IGSS preparations can be observed indefinitly. and can be used on tissue with natural fluorescence such as monocotyledonous epidermis, which are rich in diphenolic compounds. or on young lignified tissues. Moreover. the immunogold staining techniques present the opportunity for the observation of staining reactions both at the light and the electron microscope levels. As IGSS staining can be observed with any optical microscope, it is a more accessible technique than immunofluorescence. l’ransport
of gold
antibody
complexes
and wall
porosity.
The work reported here shows that wall permeabilisation allows not only the penetration of tluorochromelantibody conjugates as shown by Lloyd ( 1979) and Wick et (11.(1981). but also 5 nm-gold/antibody complexes. However. wall pore size is reported to be approximately 4.0 nm or less (Tepfer & Taylor 1981; Baron-Epel et ~11. 1988). It is clear from these data and from other works (Lloyd et ul. 1979: Doonan er (11.1984) that wall permeabilisation is a necessarystep to allow penetration of antibody complexes. There are two possible explanations for the changes in wall structure which permit the passageof gold-conjugated antibodies: either the permeabilisation process may enhance wall porosity by enlarging the trans-wall channels, or the basic cell wall structure is destroyed. The ultrastructure of epidermal walls after enzymatic treatment for 8 min is revealed by the PATAg test (Figs. 5 & 6). The walls exhibit a slight layering in the inner zone (youngest) while the external zone (oldest) is randomly organised. In the layered area. arced patterns can be detected. These descriptions recall those made in previous studies where. under the same conditions of fixation. controlled extraction of wall components reveals organised cellulosic framework especially in the inner part of the wall. The arced patterns observed in sections are characteristics of a helicoidal
Cell Biology
international
Reports,
Vol. 14, No. 12. December
1990
Ultrustructtrre 0 f pertneubilised cell wull. Fic.5. Epidcnnul &I.K The inner pan eflhe rc*all erhihir.v lu!vrin,g \c*hil.vr rhp oltrer pun is rundomly orgunixd. Arced patterns urc ourlined (P.A. T. A,q .vruininx). (DW) rli.w~~uni.wd urw: llA WI layered ureu: (pm I plumrclt,lc~tnhrarle: ((ye) clrliclp. X 2 7 m. of rhe Iuwrcd inner pun of an epidcrtnul w/l. X 35 m. Fi q. 6. High twqnifkutinn
1116
Ceil Biology
international
Reports, Vol. 14, No. 12, December
1990
arrangement (Roland & Vian. 1979: Roland it al.. 1987). as often encountered in epidermal cell walls. Since the fixation conditions for light and electron microscopy differ. it is difficult to draw a precise conclusion regarding the mode of penetration of the antibody-gold complex. However, these data suggest that the penetration of the gold/conjugate is due to an overall increase in wall porosity rather than occurrence of random “holes” through the wall. After lo-12 min of enzyme treatment, basic wall structure is not recognisable (results not shown), therefore the accessibility to antigenic sites in the cells is increased but integrity of the cell shape is altered. Cell wall porosity is supposed to be controlled “by the pectin network or by hemicelluloses (Baron-Epel et al. 1988; Mac Cann er al. 1990). These authors show that removal of pectins by pectinase or solvent greatly increases the wall porosity. Therefore, as the usual permeabilising enzymatic solutions could affect cell shape or microtubule integrity (Lee et al. 1989) it would be useful to perform wall permeabilisation by using specific pectinase or hemicellulase mixtures. Use of organic solvents such as methylamine to extract matricial wall components without damageto the basic framework of the wall could be also employed (Satiat-Jeunemaitre. unpublished). ACKNOWLEDGEMENTS: I wish to thank Professor A.M. Lambert and Dr A.C. Schmit (Universite Louis Pasteur, Strasbourg), for introducing me to the immunogold techniques used in this paper, and Dr J. Traas and C. Lloyd for the immunofluorescence ones. Thanks are also due to Dr C.R. Hawes (School of Biological and Molecular Sciences, Oxford Polytechnic, U.K.) for the use of Electron Microscopy facilities and for critically reading the manuscript.
REFERENCES: Bajer, AS., Sato, H. and Mok-Rajer, .I. (1986). Video microscopy of colloidal gold particles and immunogold labelled microtubules in improved rectified DIC and epi-illumination. Cell Struct. funct., 11: 3 17-330. P.K. and Schindler, M. (1988). Pectin as Baron-Epel, O., Gharyal, mediators of wall porosity in soybean cells. Planta. 175: 389-395. M. and De De Mey, J., Lambert, A.M., Rajer, AS., Moremans, Rrahander, M. (1982). Visualization of microtubules in interphase and mitotic plant cells of Haemanthus endosperm with the immunogold staining method. Proc. Natl. Acad. Sci., U.S.A., 79: 1898-1902. Dansher, G. and Niirgaard, J.O.R. (1983). Light microscopic visualization of colloidal gold on resin-embedded tissue. J. Histochem. Cytochem., 31, 1394- 1398. Doonan, J. II., Cove, D.J. and Lloyd, C.W. (1984). Brief cellulase treatment permits antitubulin staining of an entire filamentous organism (Moss). In: 1. Potrykus, C.T. Kinnen, A.R. Hutter, P.J. King, R.D. Shillito: Protoplasts 1983. Poster proceedings. Birkhauser Verlag. Basel, Boston, Stuttgart 1984.
Cell Biology
international
Reports,
Vol. 14. No. 12, December
1990
1117
Franke, W., Seih, E., Oshorn, M., Weher, K., Herth, W. and Falk, H. (1977). Tubulin-containing structures in the anastral mitotic apparatus of endosperm cells of the plant Leucogum aestivum as revealed by immunofluorescence. Cytobiol.,
15: 24-48. Holgate, C.S., Jackson,
P., Cowen,
P.N. and Bird,
C.C. (1983).
Immunogold-silver staining: new method of immunostaining with enhanced sensitivit . J. Histochem. C tochem., 31: 938-944. Iwata, d and Hogetsu, i (1988). Arrangement of cortical microtubules in avena colioptiles and mesoco*&lsand pisum epicotyls. Plant Cell Physiol., 29: 807815. Lee, N., Wetzstein, H. and Bornman, C.H. (1989). Cortical microtubule organization in Vitis protoplasts as affected by concentration of enzyme isolation medium and duration of incubation. Physiol. Plant., 77: 27-32. Lloyd, C.W. (1987). The plant cytoskeleton: the impact of fluorescence microscop . Ann. Rev. Plant Physiol., 38: 119-221. Lloyd, c! .W., Slabas, A.R., Powell, A.J. and Lowe, S.B. (1980). Microtubules, protoplasts and plant cell shape. An immunofluorescent study. Planta,
147: 500-506. Lloyd, C.W., Slabas, AR.,
Powell, A.J. and Mac Donald, G. (1979).
Cytoplasmic microtubules of higher plant cells visualized with anti-tubuhn antibodies. Nature, 279: 239-24 1. I,loyd, C.W. and Wells, B. (1985). Microtubules are at the tips of root hairs and form helical patterns corresponding to inner wall fibrils. J. Cell Sci., 75: 225-238. Mac Cann, M.C., Wells, B. and Roberts, K. (1990). Direct visualization of cross-links in the primary plant cell wall. J. Cell Sci., 96: 323-334.
Morejohn, L.C., Bureau, T.E., Mole-Bajer, J., Bajer, A.S. and Fosket, DE (1987). Oryzalin, a dinitroaniline herbicide, binds to plant tubulin and
inhibits microtubule polymerization in vitro. Planta, 172: 252-264. Roberts, I.N., Lloyd, C.W. and Roberts, K. (1985). Eth lene-induced microtubule reorientations: mediation by helical arrays. Planta, 164: 4K9447.
Roland, J.C., Reis, D., Vian, B., Satiat-Jeunemaitre, B. and Mosiniak, M. (1987). Morphogenesis of plant cell walls at the supramolecular level: internal geometry and versatility of helicoidal expression. Protoplasma, 140: 75-91. Roland, J.C. and Vian, B. (1979). The wall of the growing plant cells: its three dimensional organization. Int. Rev. Cytol., 61: 129-166. Satiat-Jeunemaitre, B. (1989). Microtubules, microfibrilles par&ales et morphogentse vt &ale: ces des cellules en extension. Bull. Sot. trot. Fr., 136, Actual. bot.: 87-9 i . Simmonds, D.H., Seagull, R.W. and Setterfield, G. (1985). Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells. J. Histochem. Cytochem., 33: 345-352. Smith-Huerta, N.L. and Jernstedt, J.A. (1989). Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy. Protoplasma, 151: l-10. Tepfer, M. and Taylor, I.E.P. (1981). The permeability of plant cell walls as measured by gel fiRrahon chromatography. Science, 213: 761-763. Thiery, J.P. (1967). Mise en evidence des pal saccharides sur coupes fines en microscopic Clectronique. J. Microsc. 6: 987- 1011.
Traas, J.A., Braat, P., Emons, A.M., Meekes, H. and Derksen, J.W. (1985). Microtubules in root hairs. J. Cell Sci., 76: 303-320. Van der Valk, P., Rennie, P.J., Connoly, J.A. and Fowke, L.C.
1118
Cell Biology
International
Reports,
Vol. 14. No. 12. December
1990
(1980). Distribution of cortical microtubules in tobacco protoplasts. An immunofluorescence microscopic and ultrastructural study. Protoplasma, 105: 27-43.
Weher, K., Oshorn, M., Franke, W., Seib., IL, Scheer, U. and Herth, W. (1977). Identification of molecular structures in diverse plant and animal cells by immunological cross-reaction revealed in immunofluorescence microscopy using antibody against tubulin from porcine brain. Cytobiol., 15: 285-302.
Wick, S.M., Seagull, R.W., Oshorn, M., Weber, K. and Gunning, B.E.S. (1981). Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J. Cell Biol., 89: 685690. Paper
received
21.08.90.
Revised
paper
accepted
01.10.90.
International
Reports,
Vol. 14, No. 12, December
1990
1109
VISIJAI,IZATION OF MICROTURUIXS IN WAI,IXD PI,ANT
0309-l
651/90/l
21109-l
O/SO3.00/0
0 1990 Academic Press Ltd
1110
Cell Biology
international
Reports,
Vol. 14, No. 12. December
1990
enhancement procedure (IGSS) based on the techniques developed by Dansher and Niirgaard (1983) and Holgate et al. (1983). In order to visualise and to discuss the cell wall modifications induced by the enzymatic permeabilisation, cell wall ultrastructure has been checked by electron microscopy after polysaccharide staining. MATERIAI,
AND METHODS:
Plant material. Seedsof mung bean (Vigna rdiatu) were immersed for 4 h in distilled water, and allowed to germinate on moistened vermiculite in plastic boxes at 26 C in darkness. When the shoots were 3 days old, strips from the growing part of the hypocotyl (just below the hook) were pealed as described below. Preparations of tissues for whole cell immunocytochemistry. Strips of epidermal cells were pealed from the hypocotyls under fixative for 45 min in freshly prepared 3% paraformaldehyde in microtubule-stabilising buffer (MSB), pH 6.8 (MSB = 100 mM PIPES, 10 mM EGTA, 5 mM MgS04). Strips were then immersed for 15 min in 1% NaBH4 in MSB in order to saturate free aldehyde groups and washed for 45 min in MSB. A partial cell wall digestion was performed by an 8-10 min treatment with 2% cellulase (Sigma) and 1% macerozyme R-10 (Yakult biochemicals co.) in MSB, followed by a 30 min wash in MSB. Preservation of cellular shapewas checked in a few specimens after staining with Calcofluor white. A 10 min immersion in 1% Nonidet NP40 (Sigma) in MSB was performed to permeabilise the plasma membrane. Strips were washed again in MSB, teased flat onto coverslips with needles and allow to air dry overnight. A progressive and perfect air drying was generally enough to achieve satisfactory adhesion of the strips onto the coverslips. For immunofluorescence observations, adhesion was improved by prior treatment of the coverslips with a 5 mg/ml poly-L-lysine solution (MW > 300 000, Sigma) (coverslips were left in contact with the poly-L-lysine solution for 3 h and then washed in distilled water). The slides were used either wet or dry. Immunocytochemistry. The immunocytochemical reaction was adapted from the protocols of Lloyd and Wells (1985) and Simmonds et al. (1985) as follows: in order to reduce background staining preparations were first incubated for 15 min in normal serum of the productive wies of the second antibody. The primary antibody (monoclonal antibodies to tubulin) was applied for 3-12 h at 26 C, followed by 1 h washing in 1% fish gelatin (Sigma) in MSB. Preparations were then incubated for 1 h at 26 C in darkness with a second antibody conjugated to a fluorescent probe (FITC or Texas red) or to 5 nm gold particles depending on the method of visualisation. Several ways of microtubule detection were used in order to spot differences in microtubule organisation: Immunofluorescence: a) Rat monoclonal antibodies to a tubulin at 1:15 dilution (Sera Lab distributed by Biosys, France), revealed by FITC rabbit anti-rat antibodies at 1:4OO(Biosys). b) Mouse monoclonal antibodies to @tubulin at 1:400 (Amersham) revealed either by FITC-conjugated sheep anti mouse antibodies at 1: 150 dilution or by Texas redconjugated sheepanti mouse antibodies at 1:80 dilution (Amersham).
Cell Biology
International
Reports,
Vol. 14, No. 12. December
1990
1111
Immunogold staining: Mouse monoclonal antibodies to B tubulin at 1:400 (Amersham) and secondly layer of 5 nm gold conjugated goat anti-mouse serum at 1:15 dilution (Amersham). I,ight Microscopy. Slides prepared for fluorescence microscopy were washed in I % fish gelatin in MSB for 1 h. and mounted in Citifluor (City University. London). They were observed with a Zeiss Axiophot epifluorescnece microscope. with specific FITC filter set or rhodamine filter set for Texas red. Slides prepared for IGSS were washed with filtered distilled water. The gold label was silver enhanced according to the technique of Dansher er al. (1983) with the Janssenintense 2 kit. Preparations were then mounted in Eukitt. Micrographs were taken on Kodak Thlax 400 or Ilford HP5 films. Electron microscopy and cell wall staining. The techniques used were adapted from Roland and Vian ( 1979). Strips of epidermal cells were pealed from the upper part of the hypocotyl (exponential growing area area. below the hook). Specimens were fixed in 4 % glutaraldehyde in 0.1 M sodium cacodylate buffer. pH 6.8, and washed in the samebuffer. A partial cell wall digestion was performed by an 8- 10 min treatment in enzymatic mixture as for light microscopv described above. followed by a 30 min wash in buffer. Specimens were dehydrated in a graded water/alcohol series and embedded in Spurr resin. In order to visualize the cell wall texture. ultrathin sections were treated with periodic acid/thiosemicarbazide/silver proteinate. (P.A.T.Ag). the cytochemical test for polysaccharides (Thiery 1967). Preparations were examined with a JEOL 1200EXIl transmission electron microscope.
RESULTS
AND DISCUSSION:
Comparison of IMF and IGSS techniques. As IMF techniques are currently used to detect microtubules in permeabilised cells they have been used here as a control for the IGSS techniques. Figure 1 shows immunofluorescence staining, at the tissue level, of microtubules in hypocotyl epidermis. Although the staining is rarely homogeneous through the whole tissue, the microtubular network is well detected (cf also Fig. 1 in Roberts CTal., 1985, and Fig. 5 a-c in Satiat-Jeunemaitre, 1989). Cortical microtubules appear organ&d in parallel arrays around the cells, in transverse, oblique or longitudinal directions according the cells observed. In Figure 2, a similar microtubular network is observed by immunogold staining after silver enhancement (see also Figs 5 d-f in SatiatJeunemaitre, 1989). It appears from these observations that the non-specific background staining is less of a problem with the immunogold staining technique than with immunofluorescence. Closer observations on microtubular arangements were performed on isolated cells (Figs. 3 & 4). Whatever the immunostaining combination used, the pattern of microtubule distribution was similar. There are no differences between staining with complexes directed against alpha- or beta-tubulin (cf Figs. 1 & 3 or 3 & 4). Again, IGSS and IMF techniques give similar information on the microtubular cytoskeleton:
1112
Cell Biology
International
Reports,
Vol. 14, No. 12, December
I990
Cell Biology
International
Reports,
Vol. 74, No. 12, December
1990
1113
1114
Cell Biology International
Reports, Vol. 14, No, 12, December
7990
Compurison of luhellin~ hg IMF or IGSS on isoluted walled cells. FiG. 3. (unti-atuhulin, FITCH. A honnqeneous and re~nlar urrun~etnent of tnicrotuhules uround the cell is revealed. Diflerent.focal planes (A und Bi .sholr* thur tnicrotuhules are arrunxed in purullel and continotts trun.s\‘er.se urruy. Note that both anti-hew und -ulphu tuhulin give sitnilar stainin~s puttems. X 650. Firr.4. Helical arrangewent of microtubules around a cell is revealed by \wiutions in .focal plunes. The continuity of rnicrotubule arruys,Potn the superior plune (A) to the inferior plune (B) of the cell.follows an helix. X 720. _____---________________________________-----------------------------------------------------~-------~-
different focal planes show that the cortical microtubules roll around the cells (Figs. 3 & 4) in parallel arrays. Where the microtubule lay in more oblique directions (Fig. 4). variations in the focal plane reveal a helical arrangement of the cortical microtubules. Since Lloyd’s first descriptions on microtubules in walled plant cells. many reports have shown that microtubules occur in stable helices in many growing cells ( Traas et ul. 1985; Roberts et al. 1985: Smith-Huerta & Jemstedt 1989: see Iwata & Hogetsu 1988, for more references).As they give similar results, either the IMF or IGSS techniques can be used in microtubule studies according the tissues studied or the aim of the observation. At the LM level, in walled ceils, IMF allows double staining with the use of different fluorochromes. but has two major disadvantages: firstly fluorochromes are sensitive to light and fade quite rapidly during observation. and secondly they must be avoided in plant tissues showing any autofluorescence. IGSS techniques obviate these two problems. IGSS preparations can be observed indefinitly. and can be used on tissue with natural fluorescence such as monocotyledonous epidermis, which are rich in diphenolic compounds. or on young lignified tissues. Moreover. the immunogold staining techniques present the opportunity for the observation of staining reactions both at the light and the electron microscope levels. As IGSS staining can be observed with any optical microscope, it is a more accessible technique than immunofluorescence. l’ransport
of gold
antibody
complexes
and wall
porosity.
The work reported here shows that wall permeabilisation allows not only the penetration of tluorochromelantibody conjugates as shown by Lloyd ( 1979) and Wick et (11.(1981). but also 5 nm-gold/antibody complexes. However. wall pore size is reported to be approximately 4.0 nm or less (Tepfer & Taylor 1981; Baron-Epel et ~11. 1988). It is clear from these data and from other works (Lloyd et ul. 1979: Doonan er (11.1984) that wall permeabilisation is a necessarystep to allow penetration of antibody complexes. There are two possible explanations for the changes in wall structure which permit the passageof gold-conjugated antibodies: either the permeabilisation process may enhance wall porosity by enlarging the trans-wall channels, or the basic cell wall structure is destroyed. The ultrastructure of epidermal walls after enzymatic treatment for 8 min is revealed by the PATAg test (Figs. 5 & 6). The walls exhibit a slight layering in the inner zone (youngest) while the external zone (oldest) is randomly organised. In the layered area. arced patterns can be detected. These descriptions recall those made in previous studies where. under the same conditions of fixation. controlled extraction of wall components reveals organised cellulosic framework especially in the inner part of the wall. The arced patterns observed in sections are characteristics of a helicoidal
Cell Biology
international
Reports,
Vol. 14, No. 12. December
1990
Ultrustructtrre 0 f pertneubilised cell wull. Fic.5. Epidcnnul &I.K The inner pan eflhe rc*all erhihir.v lu!vrin,g \c*hil.vr rhp oltrer pun is rundomly orgunixd. Arced patterns urc ourlined (P.A. T. A,q .vruininx). (DW) rli.w~~uni.wd urw: llA WI layered ureu: (pm I plumrclt,lc~tnhrarle: ((ye) clrliclp. X 2 7 m. of rhe Iuwrcd inner pun of an epidcrtnul w/l. X 35 m. Fi q. 6. High twqnifkutinn
1116
Ceil Biology
international
Reports, Vol. 14, No. 12, December
1990
arrangement (Roland & Vian. 1979: Roland it al.. 1987). as often encountered in epidermal cell walls. Since the fixation conditions for light and electron microscopy differ. it is difficult to draw a precise conclusion regarding the mode of penetration of the antibody-gold complex. However, these data suggest that the penetration of the gold/conjugate is due to an overall increase in wall porosity rather than occurrence of random “holes” through the wall. After lo-12 min of enzyme treatment, basic wall structure is not recognisable (results not shown), therefore the accessibility to antigenic sites in the cells is increased but integrity of the cell shape is altered. Cell wall porosity is supposed to be controlled “by the pectin network or by hemicelluloses (Baron-Epel et al. 1988; Mac Cann er al. 1990). These authors show that removal of pectins by pectinase or solvent greatly increases the wall porosity. Therefore, as the usual permeabilising enzymatic solutions could affect cell shape or microtubule integrity (Lee et al. 1989) it would be useful to perform wall permeabilisation by using specific pectinase or hemicellulase mixtures. Use of organic solvents such as methylamine to extract matricial wall components without damageto the basic framework of the wall could be also employed (Satiat-Jeunemaitre. unpublished). ACKNOWLEDGEMENTS: I wish to thank Professor A.M. Lambert and Dr A.C. Schmit (Universite Louis Pasteur, Strasbourg), for introducing me to the immunogold techniques used in this paper, and Dr J. Traas and C. Lloyd for the immunofluorescence ones. Thanks are also due to Dr C.R. Hawes (School of Biological and Molecular Sciences, Oxford Polytechnic, U.K.) for the use of Electron Microscopy facilities and for critically reading the manuscript.
REFERENCES: Bajer, AS., Sato, H. and Mok-Rajer, .I. (1986). Video microscopy of colloidal gold particles and immunogold labelled microtubules in improved rectified DIC and epi-illumination. Cell Struct. funct., 11: 3 17-330. P.K. and Schindler, M. (1988). Pectin as Baron-Epel, O., Gharyal, mediators of wall porosity in soybean cells. Planta. 175: 389-395. M. and De De Mey, J., Lambert, A.M., Rajer, AS., Moremans, Rrahander, M. (1982). Visualization of microtubules in interphase and mitotic plant cells of Haemanthus endosperm with the immunogold staining method. Proc. Natl. Acad. Sci., U.S.A., 79: 1898-1902. Dansher, G. and Niirgaard, J.O.R. (1983). Light microscopic visualization of colloidal gold on resin-embedded tissue. J. Histochem. Cytochem., 31, 1394- 1398. Doonan, J. II., Cove, D.J. and Lloyd, C.W. (1984). Brief cellulase treatment permits antitubulin staining of an entire filamentous organism (Moss). In: 1. Potrykus, C.T. Kinnen, A.R. Hutter, P.J. King, R.D. Shillito: Protoplasts 1983. Poster proceedings. Birkhauser Verlag. Basel, Boston, Stuttgart 1984.
Cell Biology
international
Reports,
Vol. 14. No. 12, December
1990
1117
Franke, W., Seih, E., Oshorn, M., Weher, K., Herth, W. and Falk, H. (1977). Tubulin-containing structures in the anastral mitotic apparatus of endosperm cells of the plant Leucogum aestivum as revealed by immunofluorescence. Cytobiol.,
15: 24-48. Holgate, C.S., Jackson,
P., Cowen,
P.N. and Bird,
C.C. (1983).
Immunogold-silver staining: new method of immunostaining with enhanced sensitivit . J. Histochem. C tochem., 31: 938-944. Iwata, d and Hogetsu, i (1988). Arrangement of cortical microtubules in avena colioptiles and mesoco*&lsand pisum epicotyls. Plant Cell Physiol., 29: 807815. Lee, N., Wetzstein, H. and Bornman, C.H. (1989). Cortical microtubule organization in Vitis protoplasts as affected by concentration of enzyme isolation medium and duration of incubation. Physiol. Plant., 77: 27-32. Lloyd, C.W. (1987). The plant cytoskeleton: the impact of fluorescence microscop . Ann. Rev. Plant Physiol., 38: 119-221. Lloyd, c! .W., Slabas, A.R., Powell, A.J. and Lowe, S.B. (1980). Microtubules, protoplasts and plant cell shape. An immunofluorescent study. Planta,
147: 500-506. Lloyd, C.W., Slabas, AR.,
Powell, A.J. and Mac Donald, G. (1979).
Cytoplasmic microtubules of higher plant cells visualized with anti-tubuhn antibodies. Nature, 279: 239-24 1. I,loyd, C.W. and Wells, B. (1985). Microtubules are at the tips of root hairs and form helical patterns corresponding to inner wall fibrils. J. Cell Sci., 75: 225-238. Mac Cann, M.C., Wells, B. and Roberts, K. (1990). Direct visualization of cross-links in the primary plant cell wall. J. Cell Sci., 96: 323-334.
Morejohn, L.C., Bureau, T.E., Mole-Bajer, J., Bajer, A.S. and Fosket, DE (1987). Oryzalin, a dinitroaniline herbicide, binds to plant tubulin and
inhibits microtubule polymerization in vitro. Planta, 172: 252-264. Roberts, I.N., Lloyd, C.W. and Roberts, K. (1985). Eth lene-induced microtubule reorientations: mediation by helical arrays. Planta, 164: 4K9447.
Roland, J.C., Reis, D., Vian, B., Satiat-Jeunemaitre, B. and Mosiniak, M. (1987). Morphogenesis of plant cell walls at the supramolecular level: internal geometry and versatility of helicoidal expression. Protoplasma, 140: 75-91. Roland, J.C. and Vian, B. (1979). The wall of the growing plant cells: its three dimensional organization. Int. Rev. Cytol., 61: 129-166. Satiat-Jeunemaitre, B. (1989). Microtubules, microfibrilles par&ales et morphogentse vt &ale: ces des cellules en extension. Bull. Sot. trot. Fr., 136, Actual. bot.: 87-9 i . Simmonds, D.H., Seagull, R.W. and Setterfield, G. (1985). Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells. J. Histochem. Cytochem., 33: 345-352. Smith-Huerta, N.L. and Jernstedt, J.A. (1989). Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy. Protoplasma, 151: l-10. Tepfer, M. and Taylor, I.E.P. (1981). The permeability of plant cell walls as measured by gel fiRrahon chromatography. Science, 213: 761-763. Thiery, J.P. (1967). Mise en evidence des pal saccharides sur coupes fines en microscopic Clectronique. J. Microsc. 6: 987- 1011.
Traas, J.A., Braat, P., Emons, A.M., Meekes, H. and Derksen, J.W. (1985). Microtubules in root hairs. J. Cell Sci., 76: 303-320. Van der Valk, P., Rennie, P.J., Connoly, J.A. and Fowke, L.C.
1118
Cell Biology
International
Reports,
Vol. 14. No. 12. December
1990
(1980). Distribution of cortical microtubules in tobacco protoplasts. An immunofluorescence microscopic and ultrastructural study. Protoplasma, 105: 27-43.
Weher, K., Oshorn, M., Franke, W., Seib., IL, Scheer, U. and Herth, W. (1977). Identification of molecular structures in diverse plant and animal cells by immunological cross-reaction revealed in immunofluorescence microscopy using antibody against tubulin from porcine brain. Cytobiol., 15: 285-302.
Wick, S.M., Seagull, R.W., Oshorn, M., Weber, K. and Gunning, B.E.S. (1981). Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J. Cell Biol., 89: 685690. Paper
received
21.08.90.
Revised
paper
accepted
01.10.90.