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Mycologia, 96(2), 2004, pp. 208-210.
© 2004 by The Mycological Society of America

Cell biology/ultrastructure

Scanning electron microscopy of mitotic nuclei and chromosomes in filamentous fungi


Dai Tsuchiya

     Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan

Hironori Koga

     Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi, Ishikawa 921-8836, Japan

Masatoki Taga 1

     Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 

A new method for scanning electron microscopy (SEM) of fungal mitotic nuclei and chromosomes was established for two ascomycetes, Cochliobolus heterostrophus and Neurospora crassa. Nuclei and chromosomes discharged from germling cells by the germ-tube burst method were spread on a surface of a glass slide. The spreads were impregnated with osmium-thiocarbohydrazide for conductive staining, followed by coating with platinum, and observed by field-emission SEM. Ultrastructure of fungal chromosomes and nuclei was visualized by SEM for the first time.

Key words: Cochliobolus heterostrophus, germ-tube burst method, mitosis, Neurospora crassa


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Analysis of the ultrastructure of nuclei and chromosomes using scanning electron microscopy (SEM) is interesting not only because of the novelty but also because this method contributes to solving important questions. For example, SEM makes it possible to determine whether fungal nuclei and chromosomes have structures distinct from those of plants and animals (for which ample data from SEM have been accumulated; Sumner 1991Go, Martin et al 1996Go, Iwano et al 1997Go, Takayama et al 1996Go, Martorell et al 2000Go, Wanner and Formanek 2000Go). In addition, three-dimensional information from SEM might shed light on the manner of chromatin compaction into condensed chromosomes during nuclear division in fungi.

We developed a SEM method for observing fungal mitotic nuclei and chromosomes using two ascomycetes: Cochliobolus heterostrophus (Drechs.) Drechs. (anamorph, Bipolaris maydis [Nishikado & Miyake] Shoem.) and Neurospora crassa Shear & Dodge. These species were chosen because the methods for preparing spread specimens of their nuclei and chromosomes already had been established for light microscopic observation in our laboratory (Tsuchiya and Taga 2001Go, Kato 2003Go), and we expected that with modest modifications those methods were usable for SEM as well. Experimental procedures and some observations are described below.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
Preparation of the spreads of mitotic chromosomes and nuclei. – The germ-tube burst method (Shirane et al 1988Go, Taga et al 1998Go, Tsuchiya and Taga 2001Go) was used with modifications. For C. heterostrophus strain C4 (supplied by O.C. Yoder, described in Leach et al 1982Go), a 200 µl droplet of conidial suspension (1.5 x 105/ml) containing 30 mM hydroxyurea (HU) and 3% (w/v) glucose was incubated to germinate on an ordinary glass slide (76 x 26 mm, 0.8–1.0 mm thick) under humid conditions for 3 h at 25–27 C. HU was used for synchronizing nuclear division in the germ tube. After washing with water to remove HU, 200 µl of 3% glucose was added to the germinated conidia adhering to the slide and incubation was resumed for 30 min. For N. crassa strain 74-OR23-1A, a droplet of macroconidial suspension (1–1.5 x 106/ml) made in Vogel’s minimal liquid medium (Vogel 1964Go) was placed on a glass slide and incubated 6 h at 25 C to allow germination. After washing away the medium with water, germinated conidia on the slide were incubated in newly added 200 µl Vogel’s medium containing 50 µg/ml thiabendazole for more than 2 h. Thiabendazole treatment was effective in arresting nuclear division at metaphase. After completing incubation, the germlings of both fungi were rinsed with water and treated with 0.05% glutaraldehyde (buffered with 50 mM cacodylate, 2 mM MgCl2, pH 7.2) for 30 s. The slide then was immersed in the fixative (99.5% methanol : glacial acetic acid = 7:3) for 2 h at room temperature. Bursting of the germling cells occurred upon immersion in the fixative to discharge the nuclei and chromosomes from cells and spread them on the surface of glass slide. The specimens were post-fixed for at least 2 h in 2.5% glutaraldehyde (buffered with 50 mM cacodylate, 2 mM MgCl2, pH 7.2) at room temperature.

DAPI staining. – After washing away glutaraldehyde three times with cacodylate buffer (pH 7.2) and once with water, the specimens were mounted with an antifade solution (Vectashield, Vector Laboratories) containing 1 µg/ml of 4',6-diamidino-2-phenylindole (DAPI), covered with a cover slip and observed under an epifluorescence microscope to select well-spread chromosomes. Pictures of DAPI-stained fluorescent image were taken with a CCD camera (Olympus DS4040) for recording overall figures of the selected specimens and their positions on a slide.

Conductive staining, drying and coating. – After carefully removing the cover slip, mounting solution containing DAPI was washed off the slide in water. The slides are treated serially for conductive staining with 1% osmium tetroxide (buffered with 50 mM cacodylate, 2 mM MgCl2, pH 7.2) for 10 min, with saturated thiocarbohydrazide (TCH) (buffered with 50 mM cacodylate, 2 mM MgCl2, pH 7.2) for 10 min, finally with 1% osmium tetroxide for 20 min. The specimens were dehydrated through ethanol series (50–60–70–80–90–95–100%) and freeze-dried using t-butyl alcohol. Slides were cut into small pieces (ca. 5 x 5 mm), mounted on the stubs and sputter coated with approximately 3 nm of platinum.

SEM. – Observations were made with a Hitachi S-4700 field emission scanning electron microscope at 15 kv.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
The three-dimensional ultrastructure of mitotic chromosomes and nuclei free from cytoplasm were visualized for both fungi. Examples of images are shown in FIGS. 1–4.



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  		FIG. 1. Interphase nucleus of C. heterostrophus. Inset shows the same specimen stained with DAPI. A cavity in the lower right of the nucleus probably marks the position of the nucleolus. Bar = 500 nm.

FIG. 2. Prophase nucleus of C. heterostrophus. Bar = 500 nm.

FIGS. 3, 4. Metaphase chromosomes of C. heterostrophus (FIG. 3) and N. crassa (FIG. 4). Inset in each figure is the DAPI stained image and an arrow shows the same chromosome as observed by SEM. Bar = 100 nm.

 
In our hands, interphase nuclei were flattened into round shapes reminiscent of spherical nature of the structure in its original form (FIG. 1). Nucleoplasmic contents seemed to be lost except the network of ragged threads—presumably chromatin and nuclear matrix composed of protein fibers (Wanner and Formanek 2000Go). A cavity probably representing the position of nucleolus was observed in each nucleus. At presumed prophase in C. heterostrophus, slender chromosomes were evident (FIG. 2). At metaphase, overall shape of the highly condensed chromosomes was more or less cylindrical in both C. heterostrophus and N. crassa (FIGS. 3, 4). Being composed of highly condensed chromatin, the surface of the metaphase chromosome was knobby, as reported in mammals (Martorell et al 2000Go), higher plants (Iwano et al 1997Go, Wanner and Formanek 2000Go) and flatworms (Lamatsch et al 1998Go). Centromeric region distinguished by conspicuous constriction to our surprise was not detected in the metaphase chromosomes of C. heterostrohus, suggesting that centromere or kinetochore of the C. heterostrophus chromosomes is extremely small. In N. crassa, on the other hand, centromere-like constriction was found in some chromosomes (data not shown), but further study is necessary to confirm the identity of these structures.

This study represents the first report of the ultra-structure of fungal nuclei and chromosomes using SEM. The method described here should be applicable to other filamentous fungi. We believe that the addition of SEM to the repertoire of mycological techniques will be extremely beneficial to the study of fungal nuclei and chromosomes.


   ACKNOWLEDGMENTS
 
The authors thank Y. Kato for excellent technical assistance and M. Mori for many helpful discussions. Thanks also go to O.C. Yoder and H. Nakashima for providing fungal strains used in this study. This work was supported in part by a research fellowship from the Japan Society for the Promotion of Science to D. Tsuchiya.


   FOOTNOTES
 
Accepted for publication June 12, 2003.

1 Corresponding author. E-mail: mtaga{at}cc.okayama-u.ac.jp; phone: +81 86 251 8656; fax: +81 86 251 7876


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Iwano M, Fukui K, Takaichi S, Isogai A. 1997. Globular and fibrous structure in barley chromosomes revealed by high-resolution scanning electron microscopy. Chromosome Res 5:341–349.[Medline]

Kato Y. 2003. Observation of somatic nuclei and chromosomes by the germ tube burst method and their FISH analysis in Neurospora crassa [BS thesis in Japanese]. Okayama, Japan: Okayama Univ. 21 p.

Lamatsch DK, Sharbel TF, Martin R, Bock C. 1998. A drop technique for flatworm chromosome preparation for light microscopy and high-resolution scanning electron microscopy. Chromosome Res 6:654–656.[Medline]

Leach J, Lang BR, Yoder OC. 1982. Methods for selection of mutants and in vitro culture of Cochliobolus heterostrophus. J. Gen. Microbiol. 128:1719–1729.

Martin R, Busch W, Herrmann RG, Wanner G. 1994. Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy. Chromosome Res 2:411–415.[Medline]

Martin R, Busch W, Herrmann RG, Wanner G. 1996. Changes in chromosomal ultrastructure during the cell cycle. Chromosome Res 4:288–294.[Medline]

Martorell MR, Benet J, Marquez C, Egozcue J, Navarro J. 2000. Correlation between centromere and chromosome length in human male pronuclear chromosomes: ultrastructural analysis. Zygote 8:79–85.[Medline]

Shirane N, Masuko M, Hayashi Y. 1988. Nuclear behavior and division in germinating conidia of Botrytis cinerea. Phytopathology 78:1627–1630.

Sumner AT. 1991. Scanning electron microscopy of mammalian chromosomes from prophase to telophase. Chromosoma 100:410–418.[Medline]

Taga M, Murata M, Saito H. 1998. Comparison of different karyotyping methods in filamentous ascomycetes—a case study of Nectria haematococca. Mycol Res 102:1355–1364.

Takayama S, Taketani Y, Bunno K. 1996. Scanning electron microscopy of C-Banding. Zoolog Sci 13:357–364.[Medline]

Tsuchiya D, Taga M. 2001. Cytological karytyping of three Cochliobolus spp. by the germ tube burst method. Phytopathology 91:354–360.[Medline]

Vogel HJ. 1964. Distribution of lysine pathway among fungi: evolutionary implications. Am Nat 98:435–446.

Wanner G, Formanek H. 2000. A new chromosome model. J Struct Biol 132:147–161.[Medline]




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