Ultrastructure of Ascosporogenesis in Nannizzia gypsea

Size: px

Start display at page:

Download "Ultrastructure of Ascosporogenesis in Nannizzia gypsea"

Audrey Payne
5 years ago
Views:

JOURNAL OF BACTERIOLOGY, May 1975, p. 743-748 Copyright ( 1975 American Society for Microbiology Vol. 122, No. 2 Printed in U.S.A. Ultrastructure of Ascosporogenesis in Nannizzia gypsea TERRY W.

1 JOURNAL OF BACTERIOLOGY, May 1975, p Copyright ( 1975 American Society for Microbiology Vol. 122, No. 2 Printed in U.S.A. Ultrastructure of Ascosporogenesis in Nannizzia gypsea TERRY W. HILL Department of Botany, University of Florida, Gainesville, Florida Received for publication 7 February 1975 Ascosporogenesis in Nannizzia gypsea was studied by electron microscopy. Development of ascospores began with the formation of an ascus vesicle composed of two paired unit membranes. Myelin figures consisting of coiled or concentric membranes were regularly connected with the growing ascus vesicle. Both the ascus vesicle and the myelin figures possessed an electron-dense line between paired membranes, and both were stained by the periodic acid-silver methenamine technique. Invagination of the ascus vesicle about the haploid nuclei resulted in eight uninucleate prospores bounded by two concentric membranes. Spore wall material was deposited between the two membranes of the prospores, and deposition was greatest in areas of the wall overlying stacked elements of endoplasmic reticulum. A single myelin figure surrounded by a polysaccharide halo was observed in the spore. The sexual spores of the dermatophyte Nannizzia gypsea (= Microsporum gypseum) are termed ascospores and are formed within a specialized cell, the ascus. In ascosporogenesis, several daughter protoplasts (prospores) are delimited within the cytoplasm of the ascus by a pair of cytoplasmic membranes, the prospore membranes. Deposition of wall material between the prospore membranes completes the mature ascospores. This form of cytokinesis has been termed "free cell formation" and is known only in the ascus of ascomycetous fungi (7). Three mechanisms of ascospore delimitation have been described. In yeasts, the prospore membranes arise by two different methods: by growth of individual cup-shaped, double-membraned vesicles enveloping the lobes of the meiotic nucleus (12), or by fusion of cytoplasmic vesicles in situ around the four meiotic products (1). It is generally agreed that filamentous ascomycetes delimit prospores by a third method: invagination of a peripheral doublemembrane structure, the ascus vesicle (AV), about the four to eight postmeiotic nuclei (15). The origin of the AV is in dispute. This study describes ascosporogenesis in N. gypsea. Evidence indicates that in this organism coiled cytoplasmic membranes called myelin figures (MFs) are the immediate source of AV membranes and that the location of endoplasmic reticulum (ER) within the immature spore determines the pattern of subsequent wall deposition. using the hair-baiting technique of Vanbreuseghem (19). Cleistothecia were crushed under a dissecting microscope, and single ascospores were transferred with a teasing needle to Sabouraud dextrose agar plates. Reciprocal crosses were made on autoclaved soil-hair plates, and clones were separated into compatible strains and maintained on Sabouraud dextrose agar plates at 5 C. Ascocarps developed within 2 to 3 weeks after inoculation of compatible strains on oatmeal salts agar (20). For electron microscopy, blocks of agar bearing cleistothecia were cut from the medium and fixed for 1 h at 23 C in 3% glutaraldehyde, adjusted to ph 7.2 with 0.2 M cacodylate buffer. Fixation was followed by postfixation in unbuffered 1% OSO4 for 1 h at 23 C. Specimens were treated with 2% uranyl acetate in 75% ethanol for 2 h during alcohol dehydration and embedded in Spurr's resin (17). Sectioning was performed on a Porter-Blum MT2 Ultramicrotome, using a diamond knife. After being mounted on Formvarcoated copper grids, sections were post-stained for 5 to 10 min by using Reynolds' lead citrate (16). Photographs were made with a Hitachi HU-11C electron microscope operated at 75 kv. Cytochemical localization of polysaccharide was accomplished by using the periodic acid-silver methenamine (PASM) technique (11). Ultrathin sections were oxidized for 1 to 5 min in 1% periodic acid at 23 C and incubated for 20 to 25 min in staining solution at 60 C. After incubation, sections were floated on 1% Kodak photographic fixer for 15 min at 23 C. In all steps, sections were handled in polyethylene rings. Appropriate controls omitting periodate oxidation or incubation in staining solution were performed. Sections were mounted, post-stained, and observed as before. MATERIALS AND METHODS RESULTS A culture of N. gypsea was isolated from soil In meiotic prophase I, cytoplasmic compocollected in a cattle pen at the University of Florida, nents of the ascus included mitochondria, ER, 743

744 HILL ribosomes, a ribosome-free zone of exclusion, and a fl9cculent electron-transparent material (Fig. 1). PASM stain revealed the flocculent material to be polysaccharide (see Fig. 4).

2 744 HILL ribosomes, a ribosome-free zone of exclusion, and a fl9cculent electron-transparent material (Fig. 1). PASM stain revealed the flocculent material to be polysaccharide (see Fig. 4). Single-membrane-bound vesicles and vacuoles were absent at all stages observed. In more mature asci, large numbers of MFs were seen, and some were associated with pockets of the nuclear envelope (Fig. 2). Simple invaginations of the nuclear envelope also occurred. After the second meiotic division, a peripheral system of paired membranes, the AV, appeared (Fig. 3 and 4). During meiosis, the AV consisted of an incomplete sac, partially enclosing the cytoplasm and excluding most of the peripheral polysaccharide; it was regularly connected with one or more MFs (Fig. 3). The region between the two membranes of the AV was selectively stained by PASM, as was the cell wall, plasmalemma, and flocculent polysaccharide (Fig. 4). The ER, nuclear envelope, and mitochondria did not stain, but the outer layers of the MFs often did (Fig. 5). Frequently an electron-dense line was seen in the lumen of both the AV (Fig. 6) and the outer layers of the 'S. ~&~*Ps AVVk 9,, SC. i Z E.,is.4..- '* J. BACTERIOL. MF (Fig. 7). In the PASM-stained AV, the silver was preferentially deposited upon the dense line (Fig. 4). In Nannizzia, the meiotic divisions are followed by a single mitotic division. At about this time, the AV formed a continuous double-membraned peripheral vesicle that completely enclosed the four (and eventually eight) haploid nuclei and most of the cytoplasm (Fig. 8). Most of the peripheral polysaccharide was excluded. Although the four premitotic nuclei were centrally located, the eight postmitotic nuclei were peripheral, and the AV displayed indentations and discontinuities between the nuclei (Fig. 9). In more mature asci, membranes derived from the AV partially enclosed each nucleus and a portion of the cytoplasm and organelles (Fig. 10). After cytokinesis, asci contained eight prospores, each bounded by two concentric prospore membranes and free within the residual unincorporated cytoplasm (Fig. 11). MFs were only occasionally seen in prospores. The elements of unstacked ER, which before delimitation had been randomly arrayed, lay beneath the pros- ^sne~!iii L; M 7/1 \ ' M FIG. 1. Ascus nucleus in meiotic prophase I, containing a peripheral nucleolus (Nu) and the axial cores of two synaptonemal complexes (SC). In the cytoplasm are a ribosome-free zone of exclusion (ZE) and a flocculent material (Ps). The bar in Fig represents 0.2 lsm. FIG. 2. Ascus in meiotic prophase. The cytoplasm contains numerous myelin figures (MF) and mitochondria (M). The large arrow indicates an association between an MF and the nuclear envelope (NE). The small arrow indicates a simple invagination of the NE.

$;'n w *,. 's' '4 ow0 Lt * 411} a " -1.K- _ s r. s s 3 i U N >.@,. 5 5 C _ i rni v A \,s' tt 0; <i'- 8ffZ s4r.;; { > W 'WI.e 11 is I...*** 1, 4f t s* ta -A a 4S.. 9* V. w It f. NI ER E.*. E R t.$ .? 4 @ @~~,?.At.j?t.t'. ^ -~~~ Downloaded from http://jb.asm.org/.e', :, I.t I + FIG. 3. Myelin figure (MF) displaying two prominent connections to the ascus vesicle (A V).

.? 4 @ @~~,?.At.j?t.t'. ^ -~~~ Downloaded from http://jb.asm.org/.e', :, I.t I + FIG. 3. Myelin figure (MF) displaying two prominent connections to the ascus vesicle (A V).

3 . Fw. fle. l.. s.xt C*& v. #.r P WxK s w I t..*,i,s, S ffi..'; I2the.' ' O'S S1 s r g11 F "%;. ^ -.;; >, X _ rsy_e ra Mn,w_ *_ s w ^L_ * s 0_ * 5_ ^ sn *t - sf f.s S S2; s Ps ffi h 4>M.s F t.'.*. <t, * s J jo v<'s r 's. ;'n w *,. 's' '4 ow0 Lt * 411} a " -1.K- _ s r. s s 3 i U N >.@,. 5 5 C _ i rni v A \,s' tt 0; <i'- 8ffZ s4r.;; { > W 'WI.e 11 is I...*** 1, 4f t s* ta -A a 4S.. 9* V. w It f. NI ER E.*. E R ^ -~~~ Downloaded from :, I.t I + FIG. 3. Myelin figure (MF) displaying two prominent connections to the ascus vesicle (A V). This section was stained by the periodic acid-silver methenamine technique, using 5-min oxidation plus 25-min stain. N, Nucleus. FIG. 4. Ascus periphery stained by PASM. The ascus wall (A W), plasmalemma (P1), Ps, and A V are stained, whereas the cytoplasm, endoplasmic reticulum (ER), and NE are not. The arrow indicates a region where the localization of the silver deposit upon the central electron-dense line of the A V can be seen. Five-minute oxidation plus 25-min stain. FIG. 5. Myelin figure stained with PASM. Arrow indicates silver-stained layer. Five-minute oxidation plus 25-min stain. FIG. 6. Part of the AV. Arrow indicates the central electron-dense line. PASM: 1-min oxidation + 20-min stain. FIG. 7. Myelin figure with arrows indicating central electron-dense lines similar to those of the AV. FIG. 8. Ascus containing four haploid nuclei (N). The upper nucleus is undergoing mitosis. Arrows indicate the complete A V. 745 " on October 20, 2018 by guest

746 HILL J. BACTERIOL. ft'@ ~~or : ' 'r. R t V ;S Nip i*v~~~~~~~~~ <e4 'r4t%s E 44r.7' 4.~~~~~~~~ SW~~ W St SW FIG. 9. Ascus showing five of the eight postmitotic nuclei.

4 746 HILL J. BACTERIOL. ~~or : ' 'r. R t V ;S Nip i*v~~~~~~~~~ <e4 'r4t%s E 44r.7' 4.~~~~~~~~ SW~~ W St SW FIG. 9. Ascus showing five of the eight postmitotic nuclei. Large arrows indicate invaginations of the AV. Small arrows point to the free ends of the A V, which has ruptured locally. FIG. 10. Ascus showing five of the eight nuclei being enveloped by membranes derived from the A V. Arrows indicate areas where organelles and cytoplasm are being captured by the closing membranes. FIG. 11. Prospore delimited by two concentric prospore membranes (PsM). ER underlies the PsM. Arrows indicate dilations of the PsM containing electron-transparent deposits. FIG. 12. Parts of three young spores delimited by thin spore walls (SW). Some ER appears stacked (StER). FIG. 13. Ascospore with differentially thickened SW. StER underlies the thick wall areas. Arrow indicates stacked epiplasmic membranes. PASM: 5-min oxidation + 25-min stain.

Some ER cisternae below the thickened wall appeared stacked, though others remained unstacked; the wall above these membranes was usually slightly thicker than other parts of the same wall.

5 VOL. 122, 1975 ASCOSPOROGENESIS IN N. GYPSEA 747 pore membranes after delimitation but were not otherwise localized or aggregated. Spore wall material first appeared as electron-transparent deposits in dilations of the two prospore membranes (Fig. 11). More mature spores possessed a wall of fairly uniform thickness (Fig. 12). Some ER cisternae below the thickened wall appeared stacked, though others remained unstacked; the wall above these membranes was usually slightly thicker than other parts of the same wall. At later stages the spore wall was more unevenly thickened (Fig. 13). The thickest areas of the wall were underlaid by proliferated sporoplasmic membranes, and epiplasmic counterparts were frequently seen. Also evident were MFs residing beneath the thinner regions of the spore walls (Fig. 14). Each was surrounded by a flocculent halo that stained with PASM (Fig. 15). The association of an MF with polysaccharide halo was rarely observed before this stage. In the most mature spores observed, the spore wall was bordered by the two prospore membranes; the inner membrane had become the spore plasmalemma, whereas the outer membrane now separated the mature spore from the residual unincorporated cytoplasm. DISCUSSION The mechanism of ascosporogenesis in Nannizzia is similar to that which occurs in other filamentous ascomycetes (2, 5, 14, 15, 18). The prospore membranes arise from the invagination of an AV about the haploid nuclei, and the spore wall forms between the prospore membranes. There has been disagreement concerning the source of the membranes composing the AV in various fungi. They have been reported to arise / SW - Pitfrom the nuclear envelope (14), the ER (15), the nuclear envelope plus ER (2), the plasmalemma (6), and fungal mesosomes (5). In Nannizzia the connections between MFs and the growing AV, plus the similar silver-staining dense line in both, indicate that the MFs are the immediate source of AV membranes. MFs are identical to the "fungal mesosomes" reported by Gil (5) in the asci of Arthroderma. The source of MFs in the asci of Nannizzia is not clear. Frequent associations of MFs with invaginations of the nuclear envelope indicate that they may arise in the nucleus. Simple invaginations of the envelope may be the beginning of such formation. MFs and similar structures have frequently been labeled artifacts. Curgy (4) has claimed that MFs can arise from disruption of organelles by glutaraldehyde-osmium fixation. McIntyre et al. (10) report that widespread disruption of cytoplasmic membranes of mouse lymphocytes by cryoprotectants has resulted in formation of myelinoid membrane aggregations. In Nannizzia there has been no indication that MFs arise from artificial disruption of cytoplasmic membranes, and no evidence of disruption was seen in the plasmalemma, nuclear envelope, ER, or other organelles. Nor have regular connections between MFs and other organelles been observed. Association with the nuclear envelope was seen only during meiosis; if MFs are produced as a general artificial response to treatment, these associations would not be so temporally restricted. The nature of the electron-dense, silver-staining line in the MF and AV is uncertain. It may represent a glycolipid or glycoprotein membrane component. The only other study in which the PASM stain was performed on devel- ~~~~~~~~~~~ * AP~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-A ' 'th' A_";;.~,J~ ;SW SW(8 PSi.. FIG. 14. Ascospore containing a MF surrounded by polysaccharide halo (Ps). FIG. 15. Part of an ascospore showing silver staining of the Ps. PASM: 5-min oxidation + 20-min stain.

6 748 HILL oping ascospores revealed no silver staining of the delimiting membranes of Hansenula wingei (1). However, in this organism the spores are delimited by direct coalescence of cytoplasmic membranes instead of by an ascus vesicle. After formation of the AV, invagination of the membranes delimits the prospores consisting of a single nucleus plus captured organelles. The random submural arrangement of captured ER correlates with the nearly uniform deposition of wall material in the young prospore. The widest wall areas are those overlying ER cisternae. A similar observation has been reported in Saccharomyces cerevisiae (9). In more mature spores, the association between thick-walled areas and the now localized ER cisternae is striking. The arrangement of proliferated ER below the thicker portions of the growing spore wall is compatible with a role of submural ER in either the transport of wall precursors or the direction of wall deposition. Endoplasmic reticulum involvement in wall growth has been suggested in other fungi (1, 9, 13, 15), and Moore (13) has suggested that the spacing of ER cisternae may be reflected in the pattern of spore reticulations in Ascodesmis sphaerospora. The occurrence of MFs within maturing ascospores has not been previously reported, although Steirs (18) has published photographs of ascospores of Poronia punctata that contain both lomasomes and MFs. Coiled membrane figures have frequently been implicated in carbohydrate metabolism (8), and the appearance and disappearance of a polysaccharide halo around the ascospore MF is consistent with such a role. In Nannizzia, the spore MF may possess enzymes enabling it to act as a synthesizing center for storage polysaccharide or to mobilize the reserves for other metabolic needs. Thus, an indirect role in wall formation may be indicated. In conclusion, evidence indicates that the MFs observed in the asci of Nannizzia serve to donate membranes to the growing ascus vesicle, that similar MFs in the spores may be involved in carbohydrate metabolism, and that the location of endoplasmic reticulum within the maturing spore determines the pattern of spore wall deposition. ACKNOWLEDGMENTS I thank J. W. Kimbrough, J. F. Preston III, and Leland Shanor for their constructive criticism of the manuscript; I J. BACTERIOL. also thank Henry Aldrich for his criticism and advice during the course of the research. LITERATURE CITED 1. Black, S. H., and C. Gorman The cytology of Hansenula. III. Nuclear segregation and envelopment during ascosporogenesis in Hansenula wingei. Arch. Mikrobiol. 79: Carroll, G. C The ultrastructure of ascospore delimitation in Saccobolus kerverni. J. Cell Biol. 33: Cole, G. T., and H. C. Aldrich Demonstration of myelin figures in unfixed freeze-etched fungus spores. J. Cell Biol. 51: Curgy, J.-J Influence du mode de fixation sur la possibilite d'observer des structures myeliniques dans les hepatocytes d'embryons de poulet. J. Microsc. (Paris) 7: Gil, F Mesosomes: their role in the delimitation of the ascospore. Mycopathol. Mycol. Appl. 49: Greenhalgh, G. N., and H. B. Griffiths The ascus vesicle. Trans. Br. Mycol. Soc. 54: Harper, R. A Cell-division in sporangia and asci. Ann. Bot. (London) 13: Hashimoto, T., and N. Yoshida Unique membranous system associated with glycogen synthesis in an imperfect fungus, Geotrichum candidum, p In R. Uyeda (ed.), Electron microscopy, vol. 2. Proc. 6th Int. Cong. Maruzen Co., Ltd., Tokyo. 9. Lynn, R. R., and P. T. Magee Development of the spore wall during ascospore formation in Saccharomyces cerevisiae. J. Cell Biol. 44: McIntyre, J. A., N. B. Gilula, and M. J. Karnovsky Cryoprotectant-induced redistribution of intramembranous particles in mouse lymphocytes. J. Cell Biol. 60: Martino, C. de, and L. Zamboni Silver methenamine stain for electron microscopy. J. Ultrastruct. Res. 19: Moens, P. B Fine structure of ascospore development in the yeast Saccharomyces cerevisiae. Can. J. Microbiol. 17: Moore, R. T Fine structure of mycota. I. Electron microscopy of the discomycete Ascodesmis. Nova Hedwigia Z. Kryptogamenkd. 5: Oso, B. A Electron microscopy of ascus development in Ascobolus. Ann. Bot. (London) 33: Reeves, F., Jr The fine structure of ascospore formation in Pyronema domesticum. Mycologia 59: Reynolds, E. S The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: Spurr, A. R A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: Stiers, D. L Fine structure of ascospore formation in Poronia punctata. Can. J. Bot. 52: Vanbreuseghem, R Technique biologique pour l'isolement des dermatophytes du sol. Ann. Soc. Belg. Med. Trop. 32: Weitzman, I., and M. Silva-Hutner Non-keratinous agar media as substrates for the ascigerous state in certain members of the Gymnoascaceae pathogenic for man and animals. Sabouraudia 5:

Intercellular Matrix in Colonies of Candida

Intercellular Matrix in Colonies of Candida JouRNAL OF BAcTEROLOGY, Sept. 1975, p. 1139-1143 Vol. 123, No. 3 Copyright 0 1975 American Society for Microbiology Printed in U.S.A. ntercellular Matrix in Colonies of Candida K. R. JOSH, J. B. GAVN,*