FLUORESCENT STAINING OF FUNGAL NUCLEI WITH A BENZIMIDAZOL DERIVATIVE

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1 J. Cell Sci. 29,77-84(1978) 77 Printed in Great Britain Company of Biologists Limited igjs FLUORESCENT STAINING OF FUNGAL NUCLEI WITH A BENZIMIDAZOL DERIVATIVE PAUL A.LEMKE, BERTHA KUGELMAN, HIDEO MORIMOTO, ERNEST C.JACOBS AND JOHN R.ELLISON Mellon Institute and Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213, U.S.A. SUMMARY A direct staining procedure is described for fluorescence microscopy of fungal nuclei, chromosomes and mitochondria. The fluorochrome is a benzimidazol derivative (33258 Hoechst) known to bind selectively to deoxyribonucleic acid at neutral ph. The advantages f Hoechst relative to Feulgen compounds used previously to stain these structures include a greater intensity of fluorescence, the absence of fading or rapid quenching of the fluorescence, and the omission of acid hydrolysis from the procedure for removal of ribonucleic acid Hoechst has been evaluated as a nuclear stain with a yeast (Saccharomyces cerevisiae) and a filamentous fungus (At>aricus bisporus) and appears to penetrate easily vegetative cells and spores of both fungi. INTRODUCTION Improved methods for resolving structures containing deoxyribonucleic acid (DNA) in fungi by fluorescence microscopy have been developed recently (Laane & Lee, 1975; Lemke et al. 1975; Slater, 1976; Williamson & Fennell, 1975). These methods involve fluorescent dyes that are known either to act as Feulgen reagents (Laane & Lee, 1975; Lemke et al. 1975), to intercalate with double-helical DNA (Williamson & Fennell, 1975), or to bind to DNA without intercalation (Slater, 1976; Williamson & Fennell, 1975). The compound 2-[2-(4-hydroxyphenyl)-6-benzimidazol]-6-(i-methyl- 4-piperazyl)-benzimidazol-trihydrochloride, or Hoechst, is recognized to be a dye of the third type and to bind most efficiently with certain classes of constitutive heterochromatin (Hilwig & Gropp, 1972; Latt, 1976). We have examined Hoechst as a nuclear and mitochondrial stain in 2 fungi, Saccharomyces cerevisiae and Agaricus bisporus. MATERIALS AND METHODS Strains and growth media Fungal strains examined were a haploid culture of S. cerevisiae (XJB1-1B, a met-i ade-2), a diploid culture of S. cerevisiae (E280, a his-4 trp-i can' x A 4840A, OL his-5ade-2), and a strain of A. bisporus (D-26, prototrophic). The yeast strains were grown on yeast extract-peptone medium (YEP) containing 2 % Bacto-peptone, 2 % dextrose, and 1 % yeast 6 c K L 29

2 78 P. A. Lemke and others extract. Yeast cells were grown overnight in YEP at 30 C C to an approximate titre of io 8 cells per ml. Petite strains were selected from the haploid yeast (ade-2, red colour) as slow-growing strains of white colour on YEP medium containing 2 % agar, and petite phenotype was confirmed by lack of growth on YEP medium containing a non-fermentable carbon source (3 % glycerol) in place of dextrose. The diploid yeast strain was brought to sporulation on biphthalateacetate medium (Kiienzl, Tingle & Halvorson, 1974), and these cells were harvested h following inoculation. The A. bisporus strain was grown on 5 cm 1 membranes (PD 150, DuPont, Wilmington, Delaware, U.S.A.) spread upon the surface of an agar-based growth medium (Saksena, Marino, Haller & Lemke, 1976). Spores of A. bisporus were obtained from mushrooms grown commercially (Butler County Mushroom Farm, Inc., Worthington, Pa ). Staining procedure Yeast cells, freshly harvested by centrifugation (iooog) from YEP, and mushroom tissue or spores were routinely fixed in absolute isopropanol for at least 30 min prior to staining. All cells were washed at least once in distilled water, recovered by centrifugation (1000 g), and resuspended in 10 ml of water containing 50//g/ml of Hoechst (Farbwerke Hoechst AG, Frankfurt, Germany). A stock solution of Hoechst in absolute ethanol (1 mg/ml) can be stored indefinitely at 20 C and added dropwise to obtain the concentration desired for staining. Staining time varied from 3 h (yeast vegetative cells and mushroom spores) to 16 h (meiotically dividing cells), and all cells were destained in 95 % ethanol for 30 min prior to microscopic examination. Microscopic examination Cells were mounted for examination in a 65 % solution of sucrose. A Leitz Orthoplan microscope equipped for incident-light fluorescence with a Ploem vertical illuminator was used. The light source was an Osram superpressure xenon lamp (XBO 75W/z) and the power supply was an Optiquip 1020 unit. Conditions for microscopic examination were determined from the known emission and excitation frequencies of Hoechst bound to DNA at neutral ph (Latt & Wohlleb, 1975); this spectrum indicates a maximum emission at 470 nm with about a 45-fold increase influorescenceover free Hoechst. The microscope was set at position 1 with the following: one excitation filter (UG-i), one dichroic mirror (K 400), and a barrier filter (K 420). Afluoriteobjective (95 x, 1.32 NA) was used. Photographs were taken on Adox 135-KB14 film (Fotokemika, Zagreb, Yugoslavia) with a 20-s exposure time and processed with Diafine developer (Acufine, Inc., Chicago, Illinois, U.S.A.). RESULTS AND DISCUSSION The effective use of Hoechst to stain DNA-containing structures in the yeast Saccharomyces cerevisiae is shown in Figs. 1 and 2. Cells depicted in these 2 figures represent, respectively, those of a haploid yeast strain and a meiotically dividing yeast strain. Dividing nuclei of haploid cells assume an elongated configuration during mitosis (Fig. 1 A), an atypical configuration for anaphase which has been described previously in yeast (Peterson & Ris, 1976) as well as in several filamentous fungi (for review see Fuller, 1976). Unfortunately, resolution with Hoechst is too poor and yeast chromosomes are too small to be counted in this configuration, but it appears that chromosomes are slightly condensed and aligned with the axis of elongation. In these cells mitochondrial DNA is evident as weakly fluorescent regions present mainly in the peripheral cytoplasm. A small percentage of yeast cells, significantly less than 1 % of the population, contain mitochondria but lack a nucleus (Fig. IB). Cells

Fluorescent staining of fungal nuclei Fig. i. Vegetative cells of S. cerevisiae stained with 33258 Hoechst.

3 Fluorescent staining of fungal nuclei Fig. i. Vegetative cells of S. cerevisiae stained with Hoechst. A, uninucleated cells containing numerous mitochondria (faint fluorescent spots) mainly in the peripheral cytoplasm; elongated nuclei (arrows) represent stages of mitosis. B, group of cells with mitochondria evident in the cytoplasm; one cell (arrow) lacks a nucleus. C, cells of a petite variant lacking mitochondria, D, dividing cell of petite variant showing total distribution of chromatin into bud. A, B, D, X 5000; c, x

P. A. Lemke and others Fig. 2. Stages of meiosis and ascospore formation in cells of S. cerevisiae stained with 33258 Hoechst.

4 P. A. Lemke and others Fig. 2. Stages of meiosis and ascospore formation in cells of S. cerevisiae stained with Hoechst. A, prophase I nuclei (arrows) with chromosomes evident as condensed fluorescent structures, x B, elongated nucleus (small arrow) of cell during anaphase I; meiotic tetrads (large arrows) in proasci. x c, nucleus during late prophase I. x D, elongated nuclei during anaphase II. x E, stages of meiosis II; paired nuclei (large arrow) prior to elongation and elongated nuclei (small arrow) of anaphase II in parallel configuration, x F, elongated nuclei of anaphase II in crossed configuration, x G, mature ascus containing 4 uninucleated ascospores. X4000. from a spontaneous segregant of petite or respiratory-deficient phenotype are shown in Fig. ic. These cells lack evidence for mitochondrial-dna when stained with Hoechst. Dividing nuclei in this petite strain often assume an elongated configuration, and nuclei in other cells may be extruded totally into a bud without apparent division (Fig. ID). In some preparations this latter phenomenon was observed repeatedly, resulting in a higher than expected frequency of progeny lacking both mitochondria

Fluorescent staining of fungal nuclei 81 Fig. 3. Vegetative cells of A. bisporus stained with 33258 Hoechst.

5 Fluorescent staining of fungal nuclei 81 Fig. 3. Vegetative cells of A. bisporus stained with Hoechst. A, branched terminal cell containing numerous mitochondria (faint fluorescent spots) and 5 elongated nuclei, x B, mycelial network showing irregular distribution of nuclei, x C-E, cells containing, respectively, 2, 3 and several nuclei per cell, x and chromosomal DNA. Another interpretation of this phenomenon is that it is an artifact of fixation. Diploid yeast cells as well as asci are evident in Fig. 2. Also present in this figure are stages of meiosis with evidence for an elongated configuration of nuclei during anaphase I (Fig. 2B). Chromosomes are seen in a condensed state during prophase I (Fig. 2 A, c) but appear to be more diffuse during subsequent stages of meiosis. Elongated configurations of dividing nuclei reappear during terminal stages of meiosis II (Fig. 2D-F). These configurations agree with models for chromosome movements during meiosis based mainly on ultrastructural analysis of non-chromosomal structures

P. A. Lemke and others Fig. 4. Cells from mushrooms of A. bisporus stained with 33258 Hoechst. A, meiotic tetrad in young basidium. x 6500.

6 P. A. Lemke and others Fig. 4. Cells from mushrooms of A. bisporus stained with Hoechst. A, meiotic tetrad in young basidium. x B, prophase I of meiosis with chromosomes evident as condensed fluorescent structures, x c, basidium with nuclei and mitochondria migrating into terminal sterigmata. x D, E, basidiospores containing, respectively, 4 and 8 nuclei, x F, portion of a thin-walled cell from internal tissue of mushroom cap containing several nuclei and elongated mitochondria (arrows) in the peripheral cytoplasm, x associated with meiosis and ascospore formation (for review see Heywood & Magee, 1976). Young asci with meiotic tetrads are shown in Fig. 2B and E and a mature 4-spored ascus is shown in Fig. 2G. Vegetative cells of the mushroom Agaricus bisporus are shown in Fig. 3. Individual cells contain several nuclei, and mitotic divisions are again represented by elongated configurations (Fig. 3 A). In these cells nuclei are irregularly distributed (Fig. 3B) and mitochondria are evident as faint fluorescent regions abundant in apical or tip cells

Fluorescent staining of fungal nuclei 83 (Fig. 3 A, c, D). In older cells nuclei may be clustered (Fig. 3E) and mitotic figures, as evidenced by elongation of nuclei, are less frequent.

7 Fluorescent staining of fungal nuclei 83 (Fig. 3 A, c, D). In older cells nuclei may be clustered (Fig. 3E) and mitotic figures, as evidenced by elongation of nuclei, are less frequent. Cells associated with mushrooms of A. bisporus are seen in Fig. 4. These cells include basidial cells at different states of maturation (Fig. 4A-C), basidiospores (Fig. 4D, E) and a cell from the cap tissue of a mushroom (Fig. 4F). A basidium containing the meiotic tetrad is seen in Fig. 4A and a young basidium containing condensed chromosomes during prophase I is shown in Fig. 4B. In Fig. 4c a binucleated basidium is evident and in this cell one nucleus and several mitochondria are seen entering the apical prongs or sterigmata. Details of the life-cycle of A. bisporus have been published elsewhere (Saksena et al. 1976) and indicate that spore number per basidium as well as the number of nuclei per spore can vary. The 2 basidiospores in Fig. 4D and E have, respectively, 4 and 8 nuclei. The cap tissue of A. bisporus is composed mainly of inflated thin-walled cells with a large central vacuole. Nuclei and mitochondria in this cell type are distributed in the peripheral cytoplasm just inside the cell wall (Fig. 4F). Mitochondria in these cells are more filamentous in appearance than mitochondria in vegetative cells. The advantages of Hoechst as a fluorescent dye for fungal structures containing DNA are basically three, and all are really refinements on methods employing other fluorescent dyes. First, Hoechst is broadly applicable as a stain for nuclei in both vegetative cells and spores following a single and simple procedure for fixation. Secondly, unlike Feulgen reagents, Hoechst binds selectively to DNA and can be used without acid hydrolysis of cells to remove ribonucleic acid. Finally, Hoechst is afluorochromeof high intensity and good resolution, and this fluorescence under the experimental conditions described does not fade. This work was supported in part by a fellowship to the Carnegie-Mellon Institute of Research from the Butler County Mushroom Farm, Inc., of Worthington, Pennsylvania 16262, U.S.A. REFERENCES FULLER, M. S. (1976). Mitosis in fungi. Int. Rev. Cytol. 45, HEYWOOD, P. & MAGEE, P. T. (1976). Meiosis in protists. Bad. Rev. 40, HILWIG, I. & GROPP, A. (1972). Staining of constitutive heterochromatin in mammalian chromosomes with a new fluorochrome. Expl Cell Res. 75, KOENZL, M. T., TINGLE, M. A. & HALVORSON, H. O. (1974). Sporulation of Saccliarowyces cerevisiae in the absence of a functional mitochondrial genome. J. Bad. 117, LAANE, M. M. & LEE, T. (1975). Examination of fungal nuclei with the Feulgen-fluorescence method. Mikroskopie 31, LATT, S. A. (1976). Optical studies of metaphase chromosome organization. A. Rev. Biophys. Bioeng. 5, LATT, S. A. & WOHLLEB, J. C. (1975). Optical studies of the interaction of Hoechst with DNA, chromatin, and metaphase chromosomes. Chromosoma 52, LEMKE, P. A., ELLISON, J. R., MARINO, R., MORIMOTO, B., ARONS, E. & KOHMAN, P. (1975J- Fluorescent Feulgen staining of fungal nuclei. Expl Cell. Res. 96, PETERSON, J. R. & Ris, H. (1976). Electron-microscopic study of spindle and chromosome movement in yeast Saccharcmryces cerevisiae. J. Cell Set. 22, SAKSENA, K. N., MARINO, R., HALLER, M. N. & LEMKE, P. A. (1976). Study on development of of Agaricus bisporus by fluorescent microscopy and scanning electron microscopy. J. Bad. 126,

8 84 P. A. Lemke and others SLATER, M. L. (1976). Rapid nuclear staining method for Saccliaromyces cerevisiae. J. Bad. 126, WILLIAMSON, D. H. & FENNELL, D. J. (1975). The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. In Methods in Cell Biology, vol. 12 (ed. D. M. Prescott), pp New York: Academic Press. {Received 16 February 1977)

THREE MITOSIS AND MEIOSIS OVERVIEW OBJECTIVES INTRODUCTION

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