- Email: [email protected]
Cell wall anchoring to cytoplasmic membrane of Candida albicans
FEMS Microbiology
Letters 125 (1995) 143-148
Cell wall anchoring to cytoplasmic membrane of Candida albicans KC. Hazen apb,*, B.W. Hazen a, M.M. Allietta a aDepartment of Pathology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA b Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA Received 5 October 1994; revision received 10 November
1994; accepted 10 November
1994
Abstract Cell wall ultrastructure of the opportunistic pathogenic yeast Candida albicans was investigated by. stereoscopic freeze-etching technique. Three wall layers were distinguishable by this technique. No clear periplasmic space was evident. Bilayer membrane invaginations were extensive. The outermost regions of the membrane invaginations were lined with thin, spine-like fibrils, which extended into the cell wall. We suggest that the fibrils along the invaginations are involved in anchoring the cell wall to the membrane. Keywords: Candida albicans; Cell wall; Freeze-etch; Ultrastructure; Membrane invaginations; Stereoscopy
1. Introduction The cell wall of human pathogenic fungi is in contact with the host and serves as a barrier against host defense products. Growth stage and conditions of culture influence cell wall (CW) composition and morphology [l]. Thin-sections prepared and stained by various methods for transmission electron microscopy @EM) have shown that the CW is composed of three to possibly eight layers, distinguished
primarily by their staining characteristics [2,3]. The outer layer, which is composed of mannoproteins, appears as a fuzzy coat that is poorly resolved by TEM techniques [ll.
* Corresponding author. Tel: l-804-924-8067; 8060; E-mail: [email protected]. 0378-1097/95/$09.50 0 1995 Federation SSDI 0378-1097(94)00490-O
fax: l-804-924-
of European
Microbiological
Resolution of cell architecture is greatly improved by rapid freezing methods. Three CW layers can be distinguished in weakly stained (uranyl acetate) and unfixed preparations of C. albicans [4]. The outer CW layer appears as a dense network of radially projecting fibrils and the inner wall layer appears contiguous with the plasmalemma [51. Kusamichi et al. [5], utilizing cryo-scanning electron microscopy, noted that the fibrils extended over 100 mn. This observation was confirmed by Hazen et al. [6] who, using the freeze-fracture method, detected fibrils as long as 300 nm. The freeze-fracture/ freeze-etch method provided unequivocal evidence of membrane invaginations and intramembranous particles (IMPS) [7]. Subsequent investigations, which involved conventional and freeze-fracture methods, have confirmed these observations [4,6,8,9]. The function of the membrane invaginations is unknown. Membrane invaginations are Societies. All rights reserved
K.C. Hazen et ul. / FEMS Microbiology
144
not seen in membrane preparations from mammalian cells or bacteria. Using stereoscopic freeze-etching, we investigated the structure of the invaginations.
2. Materials
and methods
2.1. Organism and growth conditions C. albicans isolate LGH1095 was grown in either Sabouraud glucose broth or yeast nitrogen base with 50 mM glucose at 37°C to stationary phase (24 h) as previously described [6]. The cells were harvested and washed at least three times with cold (0-2°C) deionized water. 2.2. Lyticase digestion of CW The CW was removed from washed yeast cells by lyticase (a pl-3 glucanase) digestion as described elsewhere [6] except the incubation period was increased to 2.5 h to ensure complete digestion and the cells were suspended in digest buffer (NaHPO, (0.05 M, pH 8.1)) containing lyticase (500 U/ml) and a protease inhibitor cocktail (sodium EDTA (1 mM), phenylmethanesulfonylfluoride (0.2 mM), leupeptin
Fig. 1. Appearance of the external surface of the outer membrane leaflet of C. albicans spheroplasts. Particulates of cell wall debris are visible. The tortuous system of membrane invaginations (I) is evident. In some areas, spine-like fibril structures 6) can be seen extending across the invaginations. Bar = 0.31 pm.
Letters 125 (109.7) 14% 148
(1 PM), and pepstatin A (I PM)) to which sorbitol (1 M) was added to provide osmotic stability. The spheroplasts were washed three times with digest buffer without lyticase and pelleted (1000 X g, 10 mitt). 2.3. Freeze-fracture
(-etch)
For freeze-etching, yeast cells and protoplasts were cryopressed on to a liquid helium cooled silver block. Cells were fractured, freeze-etched (1 min, Balzers AG, Liechtenstein) and rotary shadowed with platinum. Minimal distortion of CW features is obtained by rapid freezing in liquid helium and using no prefixation or cryoprotectant [5,10]. For stereoscopy, the specimens were photographed at two orientations which differed by a 10” rotation.
3. Results We have shown previously the CW of C. albicans to be composed of three discernible layers in uranyl acetate-stained preparations [6]. Freeze-etch analysis also demonstrated the presence, as in other fungi, of membrane invaginations [6], although the extent of the invaginations was not clearly established. To examine the invaginations further, we treated cells with lyticase to remove the CW (spheroplast formation) and then freeze-fractured and -etched the cells. Only unfractured but etched spheroplasts were examined. A tortuous system of membrane invaginations is evident on the outside surface (external surface of the E face) of the entire C. albicans cell membrane after spheroplast formation (Fig. 1). In some areas, fibril-like structures can be seen associated with the invaginations. These appear to extend across the invaginations. Stereoscopic examination of the invaginations from intact cells that were fractured and etched demonstrated the presence of radiating, tapered fibrils lining the perimeter of the invaginations (not shown). Further examination demonstrated that these radiating fibrils or spine-like structures emanate from the P face of the inner membrane leaflet, penetrate the outer leaflet and enter the CW (Fig. 2). The spine-like structures were found only associated with the perimeter of the invaginations and not at membrane sites between invaginations.
K.C. Hazen et al. /FEMS
Microbiology Letters 125 (1995) 143-148
Fig. 2. Stereoscopic image pairs of membrane invaginations demonstrating the spine-like structures extending through the outer membrane leaflet into the cell wall (top pair) and lining the perimeter of an invagination (middle and bottom pairs). Bar = 0.11 pm.
146
K.C. Hazen et al./FEMS
Microbiology
4. Discussion
In previous freeze-fracture studies of yeast membrane ultrastructure, yeast cells were first fixed or placed in cryoprotectant (such as glycerol) prior to slow freezing [3,9]. Such methods ultimately compromise the integrity of the yeast cell membrane [5,101. Ultrafast freezing (slam freezing) with liquid nitrogen minimizes these problems [lo]. Combined with stereoscopy, ultrafast freezing provides a powerful approach to study membrane ultrastructure and facilitated the demonstration of spine-like fibrils of invaginations. However, although these methods should reduce artifact, some degree of plastic deformation of the membrane could occur and result in the appearance of the peripheral fibrils and the invaginations [ll]. Despite this possibility, our results indicate that these membrane observations are intimately associated with the CW. The role of the invaginations in yeast physiology is unclear. Earlier studies suggested that the invaginations may be involved in cell growth. The concentration of membrane invaginations of 5. cerevisiae changes in response to growth stage and growth cycle [9]. Exponential phase cells (actively growing, constant new CW synthesis) have fewer invaginations per km2 than stationary phase cells 1121.The apical regions of growing germ tubes and hyphae of C. albicans have fewer invaginations per pm2 than older sections [3,7,13]. We found that the invaginations form a long, complex network that appears randomly arranged over the entire membrane of stationary phase yeast cells (Fig. 1). Early exponential and mid-exponential yeast cells of C. albicans appear to have short invaginations [3]. Our results are in contrast to those of Takeo [12], who reported that the membrane invaginations of S. cerevisiae are relatively short and their dimensions do not differ between exponential and stationary phase cells. Treatment with the imidazoles, lombazole, miconazole, and bifonazole, causes a decrease in the number of membrane invaginations and separation of the CW from the membrane of C. albicans [8,14]. Treatment with nystatin or filipin, which are polyenes and bind to membrane ergosterol, does not result in CW distortions or the dissociation of the wall from the membrane seen with imidazoles [12,15]. It is not clear whether these polyenes cause deformations to
Letters 125 (1995) 143-148
the invaginations [3,12]. These various observations suggest that the invaginations are involved in maintenance of CW integrity. Penetration of the spine-like structures lining the perimeter of the invaginations into the CW indicates that they allow contiguity of the invaginations with the CW. Alternatively, the spine-like structures may be poiysaccharide microfibrils synthesized at the membrane for wall incorporation or they may be involved in polysaccharide assembly. These possibilities are unlikely as chitin and glucan (the major polysaccharide components of the C. albicans CW) are synthesized at specialized sites in the membrane, which are not associated with invaginations [3,7]. The possibility that mannan synthesis occurs at the invaginations and is evident as spine-like structures cannot be dismissed. However, mannan as well as other CW polysaccharides, are predominantly synthesized during active growth when new CW is needed for emerging and expanding cells. It is at this time that the invaginations are least evident ([9] and our unpublished results). A recent study using immunoelectron microscopy demonstrated that cytoskeletal actin is associated with the cytoplasmic base of the invaginations [16]. Whether the spine-like structures emanating from the periphery of the invaginations is related to cytoskeietal actin requires further investigation. In view of the available evidence concerning the abundance of invaginations at more mature portions of a cell, the detachment of the CW from the membrane after treatment of cells with imidazoles, and the improbability that the spine-like fibrils are nascent wall polysaccharide material as described above, we suggest that the fibrils, in connection with the invaginations, may serve to deplasticize the CW. Cell wall rigidity would occur. In turn, once the anchoring effect has been established, local cell membrane fluidity may also be decreased.
Acknowledgements
We thank Dr. Thomas Tillack for helpful discussions. This work was supported by United States Public Health Service grant AI31048 (K.C.H.) from the National Institutes of Health (USA).
K.C. Hazen et al. /FEMS Microbiology Letters 125 (1995) 143-148
References [9] [l] Cassone, A. (1989) Cell wall of Candida albicans: its functions and its impact on the host. Curr. Top. Med. Mycol. 3, 248-314. [2] Poulain, D., Tronchin, G., Dubremetz, J.F. and Biguet, J. (1978) Ultrastructure of the cell wall of Candida albicans blastospores: study of its constitutive layers by the use of a cytochemical technique revealing polysaccharides. Ann. Microbiol. (Inst. Past.) 129, 140-153. [3] Rico, H., Miragall, F. and Sentandreu, R. (1985) Abnormal formation of Candida albicans walls produced by calcofluor white: an ultrastructural and stereologic study. Exp. Mycol. 9, 241-253. [4] Borgers, M. and De Nollin, S. (1974) The preservation of subcellular organelles of Can&a albicans with conventional fixatives. J. Cell Biol. 62, 574-581. [S] Kusamichi, M., Monodane, T., Tokunaga, M. and Koike, H. (19901 Influence of surrounding media on preservation of cell wall ultrastructure of Candida afbicans revealed by low temperature scanning electron microscopy. .I. Electron Microsc. 39, 477-486. [6] Hazen, K.C. and Hazen, B.W. (1992) Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans. Infect. Immun. 60, 1499-1508. [7] Moor, H. and Miihlethaler, K. (1963) Fine structure in frozen-etched yeast cells. J. Cell Biol. 17, 609-628. [8] Barug, D. and de Groot, C. (19831 Microscopic studies of
[lo] [ll]
[12]
[13]
[14]
[15]
[16]
147
Candida albicans and Torulopsis glabrata after in vitro treatment with bifonazole. Arzneim.-Forsch. 33, 538-545. Takeo, K. (1984) Lack of invaginations of the plasma membrane during budding and cell division of Saccharomyces cerevisiae and Schizosaccharotnyces pombe. FEMS Microbiol. Lett. 22, 97-100. Heuser, J.E. (1983) Procedure for freeze-drying molecules adsorbed to mica flakes. J. Mol. Biol. 169, 155-195. Steere, R.L., Erbe, E.F. and Moseley, J.M. (1980) Prefacture and cold-fracture images of yeast plasma membranes. J. Cell Biol. 86, 113-122. Takeo, K. (1985) Resistance of the stationary-phase plasma membrane of Candida albicans to filipin-induced deformation. FEMS Microbial. Lett. 27, 73-77. Takeo, K., Shigeta, M. and Takagi, Y. (1976) Plasma membrane ultrastructural differences between the exponential and stationary phases of Saccharomyces cereuisiae as revealed by freeze-etching. J. Gen. Microbial. 97, 323-329. De Nollin, S. and Borgers, M. (1974) The ultrastructure of Candida albicans after in vitro treatment with miconazole. Sabouraudia 12, 341-351. Sekiya, T. and Nozawa, Y. (1983) Reorganization of membrane ergosterol during cell fission events of Candida albicans: a freeze-fracture study of distribution of filipinergosterol complexes. J. Ultrastr. Res. 83, 48-57. Mulholland, J., Preuss, D., Moon, A., Wong, A., Drubin, D. and Botstein, D. (1994) Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J. Cell Biol. 125, 381-391.
Letters 125 (1995) 143-148
Cell wall anchoring to cytoplasmic membrane of Candida albicans KC. Hazen apb,*, B.W. Hazen a, M.M. Allietta a aDepartment of Pathology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA b Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA Received 5 October 1994; revision received 10 November
1994; accepted 10 November
1994
Abstract Cell wall ultrastructure of the opportunistic pathogenic yeast Candida albicans was investigated by. stereoscopic freeze-etching technique. Three wall layers were distinguishable by this technique. No clear periplasmic space was evident. Bilayer membrane invaginations were extensive. The outermost regions of the membrane invaginations were lined with thin, spine-like fibrils, which extended into the cell wall. We suggest that the fibrils along the invaginations are involved in anchoring the cell wall to the membrane. Keywords: Candida albicans; Cell wall; Freeze-etch; Ultrastructure; Membrane invaginations; Stereoscopy
1. Introduction The cell wall of human pathogenic fungi is in contact with the host and serves as a barrier against host defense products. Growth stage and conditions of culture influence cell wall (CW) composition and morphology [l]. Thin-sections prepared and stained by various methods for transmission electron microscopy @EM) have shown that the CW is composed of three to possibly eight layers, distinguished
primarily by their staining characteristics [2,3]. The outer layer, which is composed of mannoproteins, appears as a fuzzy coat that is poorly resolved by TEM techniques [ll.
* Corresponding author. Tel: l-804-924-8067; 8060; E-mail: [email protected]. 0378-1097/95/$09.50 0 1995 Federation SSDI 0378-1097(94)00490-O
fax: l-804-924-
of European
Microbiological
Resolution of cell architecture is greatly improved by rapid freezing methods. Three CW layers can be distinguished in weakly stained (uranyl acetate) and unfixed preparations of C. albicans [4]. The outer CW layer appears as a dense network of radially projecting fibrils and the inner wall layer appears contiguous with the plasmalemma [51. Kusamichi et al. [5], utilizing cryo-scanning electron microscopy, noted that the fibrils extended over 100 mn. This observation was confirmed by Hazen et al. [6] who, using the freeze-fracture method, detected fibrils as long as 300 nm. The freeze-fracture/ freeze-etch method provided unequivocal evidence of membrane invaginations and intramembranous particles (IMPS) [7]. Subsequent investigations, which involved conventional and freeze-fracture methods, have confirmed these observations [4,6,8,9]. The function of the membrane invaginations is unknown. Membrane invaginations are Societies. All rights reserved
K.C. Hazen et ul. / FEMS Microbiology
144
not seen in membrane preparations from mammalian cells or bacteria. Using stereoscopic freeze-etching, we investigated the structure of the invaginations.
2. Materials
and methods
2.1. Organism and growth conditions C. albicans isolate LGH1095 was grown in either Sabouraud glucose broth or yeast nitrogen base with 50 mM glucose at 37°C to stationary phase (24 h) as previously described [6]. The cells were harvested and washed at least three times with cold (0-2°C) deionized water. 2.2. Lyticase digestion of CW The CW was removed from washed yeast cells by lyticase (a pl-3 glucanase) digestion as described elsewhere [6] except the incubation period was increased to 2.5 h to ensure complete digestion and the cells were suspended in digest buffer (NaHPO, (0.05 M, pH 8.1)) containing lyticase (500 U/ml) and a protease inhibitor cocktail (sodium EDTA (1 mM), phenylmethanesulfonylfluoride (0.2 mM), leupeptin
Fig. 1. Appearance of the external surface of the outer membrane leaflet of C. albicans spheroplasts. Particulates of cell wall debris are visible. The tortuous system of membrane invaginations (I) is evident. In some areas, spine-like fibril structures 6) can be seen extending across the invaginations. Bar = 0.31 pm.
Letters 125 (109.7) 14% 148
(1 PM), and pepstatin A (I PM)) to which sorbitol (1 M) was added to provide osmotic stability. The spheroplasts were washed three times with digest buffer without lyticase and pelleted (1000 X g, 10 mitt). 2.3. Freeze-fracture
(-etch)
For freeze-etching, yeast cells and protoplasts were cryopressed on to a liquid helium cooled silver block. Cells were fractured, freeze-etched (1 min, Balzers AG, Liechtenstein) and rotary shadowed with platinum. Minimal distortion of CW features is obtained by rapid freezing in liquid helium and using no prefixation or cryoprotectant [5,10]. For stereoscopy, the specimens were photographed at two orientations which differed by a 10” rotation.
3. Results We have shown previously the CW of C. albicans to be composed of three discernible layers in uranyl acetate-stained preparations [6]. Freeze-etch analysis also demonstrated the presence, as in other fungi, of membrane invaginations [6], although the extent of the invaginations was not clearly established. To examine the invaginations further, we treated cells with lyticase to remove the CW (spheroplast formation) and then freeze-fractured and -etched the cells. Only unfractured but etched spheroplasts were examined. A tortuous system of membrane invaginations is evident on the outside surface (external surface of the E face) of the entire C. albicans cell membrane after spheroplast formation (Fig. 1). In some areas, fibril-like structures can be seen associated with the invaginations. These appear to extend across the invaginations. Stereoscopic examination of the invaginations from intact cells that were fractured and etched demonstrated the presence of radiating, tapered fibrils lining the perimeter of the invaginations (not shown). Further examination demonstrated that these radiating fibrils or spine-like structures emanate from the P face of the inner membrane leaflet, penetrate the outer leaflet and enter the CW (Fig. 2). The spine-like structures were found only associated with the perimeter of the invaginations and not at membrane sites between invaginations.
K.C. Hazen et al. /FEMS
Microbiology Letters 125 (1995) 143-148
Fig. 2. Stereoscopic image pairs of membrane invaginations demonstrating the spine-like structures extending through the outer membrane leaflet into the cell wall (top pair) and lining the perimeter of an invagination (middle and bottom pairs). Bar = 0.11 pm.
146
K.C. Hazen et al./FEMS
Microbiology
4. Discussion
In previous freeze-fracture studies of yeast membrane ultrastructure, yeast cells were first fixed or placed in cryoprotectant (such as glycerol) prior to slow freezing [3,9]. Such methods ultimately compromise the integrity of the yeast cell membrane [5,101. Ultrafast freezing (slam freezing) with liquid nitrogen minimizes these problems [lo]. Combined with stereoscopy, ultrafast freezing provides a powerful approach to study membrane ultrastructure and facilitated the demonstration of spine-like fibrils of invaginations. However, although these methods should reduce artifact, some degree of plastic deformation of the membrane could occur and result in the appearance of the peripheral fibrils and the invaginations [ll]. Despite this possibility, our results indicate that these membrane observations are intimately associated with the CW. The role of the invaginations in yeast physiology is unclear. Earlier studies suggested that the invaginations may be involved in cell growth. The concentration of membrane invaginations of 5. cerevisiae changes in response to growth stage and growth cycle [9]. Exponential phase cells (actively growing, constant new CW synthesis) have fewer invaginations per km2 than stationary phase cells 1121.The apical regions of growing germ tubes and hyphae of C. albicans have fewer invaginations per pm2 than older sections [3,7,13]. We found that the invaginations form a long, complex network that appears randomly arranged over the entire membrane of stationary phase yeast cells (Fig. 1). Early exponential and mid-exponential yeast cells of C. albicans appear to have short invaginations [3]. Our results are in contrast to those of Takeo [12], who reported that the membrane invaginations of S. cerevisiae are relatively short and their dimensions do not differ between exponential and stationary phase cells. Treatment with the imidazoles, lombazole, miconazole, and bifonazole, causes a decrease in the number of membrane invaginations and separation of the CW from the membrane of C. albicans [8,14]. Treatment with nystatin or filipin, which are polyenes and bind to membrane ergosterol, does not result in CW distortions or the dissociation of the wall from the membrane seen with imidazoles [12,15]. It is not clear whether these polyenes cause deformations to
Letters 125 (1995) 143-148
the invaginations [3,12]. These various observations suggest that the invaginations are involved in maintenance of CW integrity. Penetration of the spine-like structures lining the perimeter of the invaginations into the CW indicates that they allow contiguity of the invaginations with the CW. Alternatively, the spine-like structures may be poiysaccharide microfibrils synthesized at the membrane for wall incorporation or they may be involved in polysaccharide assembly. These possibilities are unlikely as chitin and glucan (the major polysaccharide components of the C. albicans CW) are synthesized at specialized sites in the membrane, which are not associated with invaginations [3,7]. The possibility that mannan synthesis occurs at the invaginations and is evident as spine-like structures cannot be dismissed. However, mannan as well as other CW polysaccharides, are predominantly synthesized during active growth when new CW is needed for emerging and expanding cells. It is at this time that the invaginations are least evident ([9] and our unpublished results). A recent study using immunoelectron microscopy demonstrated that cytoskeletal actin is associated with the cytoplasmic base of the invaginations [16]. Whether the spine-like structures emanating from the periphery of the invaginations is related to cytoskeietal actin requires further investigation. In view of the available evidence concerning the abundance of invaginations at more mature portions of a cell, the detachment of the CW from the membrane after treatment of cells with imidazoles, and the improbability that the spine-like fibrils are nascent wall polysaccharide material as described above, we suggest that the fibrils, in connection with the invaginations, may serve to deplasticize the CW. Cell wall rigidity would occur. In turn, once the anchoring effect has been established, local cell membrane fluidity may also be decreased.
Acknowledgements
We thank Dr. Thomas Tillack for helpful discussions. This work was supported by United States Public Health Service grant AI31048 (K.C.H.) from the National Institutes of Health (USA).
K.C. Hazen et al. /FEMS Microbiology Letters 125 (1995) 143-148
References [9] [l] Cassone, A. (1989) Cell wall of Candida albicans: its functions and its impact on the host. Curr. Top. Med. Mycol. 3, 248-314. [2] Poulain, D., Tronchin, G., Dubremetz, J.F. and Biguet, J. (1978) Ultrastructure of the cell wall of Candida albicans blastospores: study of its constitutive layers by the use of a cytochemical technique revealing polysaccharides. Ann. Microbiol. (Inst. Past.) 129, 140-153. [3] Rico, H., Miragall, F. and Sentandreu, R. (1985) Abnormal formation of Candida albicans walls produced by calcofluor white: an ultrastructural and stereologic study. Exp. Mycol. 9, 241-253. [4] Borgers, M. and De Nollin, S. (1974) The preservation of subcellular organelles of Can&a albicans with conventional fixatives. J. Cell Biol. 62, 574-581. [S] Kusamichi, M., Monodane, T., Tokunaga, M. and Koike, H. (19901 Influence of surrounding media on preservation of cell wall ultrastructure of Candida afbicans revealed by low temperature scanning electron microscopy. .I. Electron Microsc. 39, 477-486. [6] Hazen, K.C. and Hazen, B.W. (1992) Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans. Infect. Immun. 60, 1499-1508. [7] Moor, H. and Miihlethaler, K. (1963) Fine structure in frozen-etched yeast cells. J. Cell Biol. 17, 609-628. [8] Barug, D. and de Groot, C. (19831 Microscopic studies of
[lo] [ll]
[12]
[13]
[14]
[15]
[16]
147
Candida albicans and Torulopsis glabrata after in vitro treatment with bifonazole. Arzneim.-Forsch. 33, 538-545. Takeo, K. (1984) Lack of invaginations of the plasma membrane during budding and cell division of Saccharomyces cerevisiae and Schizosaccharotnyces pombe. FEMS Microbiol. Lett. 22, 97-100. Heuser, J.E. (1983) Procedure for freeze-drying molecules adsorbed to mica flakes. J. Mol. Biol. 169, 155-195. Steere, R.L., Erbe, E.F. and Moseley, J.M. (1980) Prefacture and cold-fracture images of yeast plasma membranes. J. Cell Biol. 86, 113-122. Takeo, K. (1985) Resistance of the stationary-phase plasma membrane of Candida albicans to filipin-induced deformation. FEMS Microbial. Lett. 27, 73-77. Takeo, K., Shigeta, M. and Takagi, Y. (1976) Plasma membrane ultrastructural differences between the exponential and stationary phases of Saccharomyces cereuisiae as revealed by freeze-etching. J. Gen. Microbial. 97, 323-329. De Nollin, S. and Borgers, M. (1974) The ultrastructure of Candida albicans after in vitro treatment with miconazole. Sabouraudia 12, 341-351. Sekiya, T. and Nozawa, Y. (1983) Reorganization of membrane ergosterol during cell fission events of Candida albicans: a freeze-fracture study of distribution of filipinergosterol complexes. J. Ultrastr. Res. 83, 48-57. Mulholland, J., Preuss, D., Moon, A., Wong, A., Drubin, D. and Botstein, D. (1994) Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J. Cell Biol. 125, 381-391.