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Department of Botany, PO Box 118526, University of Florida, Gainesville, Florida 32611-8526
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ABSTRACT |
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We have approached the problem of hyphal tip growth by comparing the cell wall composition of elongating and non-elongating regions of the hyphae of Achlya bisexualis. To ensure that we could distinguish between elongating and non-elongating hyphae, light microscopic observations were used to determine the rates of elongation under growing and non-growing conditions. When elongation was measured in 10 min intervals it was found to consist of fluctuating periods of fast and slow growth rates, in the form of cycles. Even under our growing conditions, however, a very small number of hyphae in a colony are not elongating. SEM analysis revealed that elongating hyphae have tapered apices, whereas non-elongating hyphae have a rounded apex. The major matrix wall components, 1,3-ß-glucans, were localized with an indirect immunogold technique specific for these polymers. This method resulted in their localization to all regions of both elongating and non-elongating hyphae, including the apex.
Key words: 1,3-ß-glucans, hyphal elongation, immunogold electron microscopy, pulse growth
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), thus raising the possibility that wall growth is also discontinuous.
In the present paper we address tip growth in Achlya and report on the distribution of the major wall component, 1,3-ß-glucans (Reiskind and Mullins 1981a
), along elongating and non-elongating hyphae. Traditionally, cell wall components have been identified cytochemically by using indirect, extractive methods. This approach can lead to problems such as incomplete extraction, and unknown effects of the extraction procedure on the ultrastructure of cell walls. Other approaches, such as enzyme-linked colloidal gold labeling, are nondestructive and can be used to localize cell wall components on thin sections (Berg et al 1988
). Colloidal gold has attained a rank of distinction among electron microscope cytochemical markers due to its versatility in adsorption to macromolecules, electron density, small size, and ability to be measured morphometrically. We have used antibodies against 1,3-ß-glucans, and an indirect labeling technique utilizing a colloidal gold complexioned with a secondary antibody, to address the distribution of these cell wall components in both elongating and non-elongating hyphal tips in Achlya. This is the first paper of an overall examination of hyphal tip growth in Achlya, where we will address the chemical composition of wall components and their ultrastructural architecture in both elongating and non-elongating hyphae.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). When the hyphae had grown about 2 to 5 mm beyond the agar plug they were ready for use in measuring hyphal elongation. Non-growing conditions were obtained by transferring growing colonies to 0.2% glucose solution (Hill and Mullins 1979
), and incubating until measurements of elongation indicated that it had stopped. These procedures made it possible to produce colonies with elongating or non-elongating hyphae prior to their chemical fixation. Screening for non-elongation is necessary, because there are a few hyphae in some colonies under non-growing conditions that are still elongating, albeit at a slow rate. Growth rate determinations – Hyphal elongation was monitored with an Olympus BH-2 light microscope using the 40x objective. The colonies were observed on depression slides with cover slips and it was possible to measure apices growing parallel to the plane of optical section. Digital images of the hyphal tips were taken every 10 min with a Pixera 120C digital camera. The microscope light was turned off between the measurements to avoid heating. Average elongation rates were calculated using a stage micrometer for calibration. A total of one hundred hyphae from different growing colonies and a total of 50 hyphae from different non-growing colonies were measured over periods of several hours.
TEM preparation – Colonies were fixed for 30 min at room temperature with 4% (v/v) glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2. After rinsing in 3 changes of buffer, the material was postfixed in 1% (w/v) osmium tetroxide in the same buffer, for 30 min. Samples were again washed several times in buffer, followed by dehydration in an ethanol series, terminating in absolute acetone.
Material in absolute acetone was infiltrated with an Epon 812 embedding medium and polymerized. Embedded samples were then thin sectioned (75–80 nm) and collected on formvar coated nickel grids. Sections were labeled by floating them face down in the antibody solution. The gold reagent and secondary antibody, 18 nm colloidal gold-affinipure goat anti-rabbit IgG, H + L, was obtained from Jackson ImmunoResearch Labs (West Grove, Pennsylvania). Two primary antibodies were used for labeling the 1,3-ß-glucans. A polyclonal antibody raised in rabbit against laminarin (purchased from Genosys Biotechnologies Inc., The Woodlands, Texas) and hereafter referred to as rabbit polyclonal, was used as described previously (Shapiro and Mullins 1997
). A monoclonal antibody raised in mouse against a laminarin-haemocyanin conjugate and hereafter referred to as mouse monoclonal, was purchased from Biosupplies Australia Pty Ltd (Parkville Victoria, Australia), and was diluted before use 1:100 in phosphate buffered saline, pH 7.2, containing 0.5% cold water fish gelatin in a 20 µL staining drop. Both of these antibodies recognize linear 1,3-ß-oligosaccharide segments in 1,3-ß-glucans, and the mouse monoclonal recognizes an epitope of at least five 1,3-ß-linked glucopyranose residues (Meikle et al 1991
). Neither antibody shows cross reactivity with 1,4-ß-glucans or mixed linked 1,3-ß-, 1,4-ß-glucans (Meikle et al 1991
, Shapiro and Mullins 1997
).
SEM preparation –
For SEM the colonies were fixed with 4% glutaraldehyde in 0.05 M sodium cacodylate buffer and washed several times in buffer. Labeling used complete submersion in the staining drop rather than floating, followed by silver enhancement, dehydrated in an ethanol series, and critical point dried. For silver enhancement the colonies were placed in a non-diluted mixture (1:1) of reagents from the Aurion Silver Enhancement Kit for 5 min, then washed several times with water to stop the reaction (Scopsi et al 1986
).
Cytochemical controls –
The following cytochemical controls were performed: (i) Pre-absorption of the primary antibody with laminarin: a 1,3-ß-linked polymer from Laminaria digitata (Sigma, St. Louis, Missouri), or with neutral and phosphorylated 1,3-ß-glucans, isolated and purified from Achlya (Lee et al 1996
). The primary antibody stock solution, 10 µL, was incubated with 100 mg of each of the polymers above in 1 mL of PBS containing 0.5% gelatin for 1 h, before it was used for labeling. (ii) Omission of the primary antibody: the standard labeling procedure was used except the incubation with primary antibody was omitted. (iii) Replacement of the primary antibody with a non-specific primary antibody: used HL 1099, raised in mouse against neurofilaments, and was provided by the Hybridoma Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, Florida).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and finally labeled. In addition, methanol fixation was used by freezing in -80 C methanol, warmed to room temperature over 2 h, then transferred into absolute acetone for TEM or rehydrated in a methanol/water series before labeling and processing for SEM. These alternative methods of fixation produced the same result as standard chemical fixation and are not reported here. On cross sections of Achlya the antibodies detected 1,3-ß-glucans in the wall and in vacuoles. In a series of sections of apical, subapical, and mature regions of the same elongating hypha (Figs. 4–10 ) strong labeling with the rabbit polyclonal antibody is present in the wall on all the sections, including the most apical area. A similar series of sections of a non-elongating hypha gave the same result (data not shown). On cross sections of Achlya the mouse monoclonal antibody gave similar results and labeled 1,3-ß-glucans in the wall and in vacuoles (Fig. 11 ). Longitudinal sections of both elongating (Fig. 12 ) and non-elongating (data not shown) hyphae treated with the mouse monoclonal antibody reveal labeling of the cell wall in the apex and all areas along the hypha. No surface labeling with mouse monoclonal or rabbit polyclonal antibodies was found in SEM preparations (Fig. 16 ). In a series of cytochemical controls pre-absorption of mouse monoclonal primary antibody with laminarin or Achlya glucans resulted in the absence of labeling (Fig. 13 ). Omission of the primary antibody resulted in the absence of the labeling (Fig. 14 ). Replacement of the primary antibody with a non-specific primary antibody raised in mouse resulted in the absence of labeling (Fig. 15 ).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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for a variety of fungal species, since they used very short time intervals (1–5 s) for measuring growth, and did not provide a basis for the pulses which were seen.
Based on the cytochemical controls (Figs. 13–15
), both antibodies used in the labeling procedure were specific for 1,3-ß-glucans, and corroborated our previous results with the same rabbit polyclonal antibody (Shapiro and Mullins 1997
). Both antibodies detect 1,3-ß-glucans in the cell wall and vacuoles. The vacuolar label identifies cytoplasmic water-soluble 1,3-ß-glucans (Lee et al 1996
, Shapiro and Mullins 1997
). The major matrix component of the wall, namely 1,3-ß-glucans, is distributed over all regions of a hypha, including the apex, in both elongating and non-elongating hyphae (Figs. 4–12
). We do not know why we did not obtain surface labeling of hyphae with SEM (Fig. 16
), since there is evidence that the 1,3-ß-glucans constitute the surface layer (Reiskind and Mullins 1981b
). It is possible that the protein component (Reiskind and Mullins 1981a
), which represents 10% of the dry weight of the wall, is present on the surface and prevents labeling. In a major contribution to the ultrastructural architecture of the walls of hyphal fungi, Hunsley and Burnett (1970)
treated hyphae with enzymes, singly or in combination, and then examined both shadow-cast and sectioned material with the electron microscope. The conditions, however, that they employed for growth very likely ensured that their cultures did not contain elongating hyphae. This is a criticism that can be applied to other papers on the subject of hyphal tip growth, where not enough attention was given to ensuring that elongation was occurring. We have used methods in the present study to ensure that either elongating or non-elongating hyphae could be obtained for indirect immunogold antibody labeling of wall 1,3-ß-glucans. Thus we conclude that the major matrix component, 1,3-ß-glucans, is present at the apex and in all other areas of the hyphal wall in both elongating and non-elongating cultures of Achlya. The next paper in this series will be directed toward determining if any other components are present at the hyphal apex under both elongating and non-elongating conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication August 7, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bourett TM, Czymmek KJ, Howard RJ., 1998 An improved method for affinity probe localization in whole cells of filamentous fungi Fungal Genet Biol 24:3-13[Medline]
Cantino EC, Horenstein EA., 1953 Carotenoids and oxidative enzymes in the aquatic Phycomycetes Blastocladiella and Rhizophlyctis Amer J Bot 40:688-694
Hill TH, Mullins JT., 1979 Hyphal tip growth in Achlya: enzyme activities in mycelium and medium Can J Bot 57:2145-2149
Hunsley D, Burnett JH., 1970 The ultrastructural architecture of the walls of some fungi J Gen Microbiol 62:203-218
Lee JH, Mullins JT, Grander JE., 1996 Water-soluble reserve polysaccharides from Achlya are beta-(1->3)-glucans Mycologia 88:254-270
López-Franco R, Bartnicki-Garcia S, Bracker CE., 1994 Pulsed growth of fungal hyphal tips Proc Natl Acad Sci USA 91:12228-12232
Meikle PJ, Bonig I, Hoogenraad NJ, Clarke AE, Stone BA., 1991 The location of (1->3)-beta-glucans in the walls of pollen tubes of Nicotiana alata using a (1->3)-beta-glucan-specific monoclonal antibody Planta 185:1-8
Reiskind JB, Mullins JT., 1981a Molecular architecture of the hyphal wall of Achlya ambisexualis Raper. I. Chemical analyses Can J Microbiol 27:1092-1099
———. 1981b Molecular architecture of the hyphal wall of Achlya ambisexualis Raper. II. Ultrastructural analyses and a proposed model Can J Microbiol 27:1100-1105
Scopsi L, Larsson L, Bastholm L, Nielsen WH., 1986 Silver enhance colloidal gold probes as markers for scanning electron microscopy Histochemistry 86:35-43[Medline]
Shapiro A, Mullins JT., 1997 Localization of cytoplasmic water-soluble reserve (1->3)-beta-glucans in Achlya with immunostaining Mycologia 89:89-91
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