Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, 332 Life Sciences, Stony Brook, NY 11794-5215, USA
Correspondence
Aaron M. Neiman
Aaron.Neiman{at}sunysb.edu
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ABSTRACT |
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INTRODUCTION |
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The spore wall is a more elaborate structure than the vegetative wall of S. cerevisiae. The four major components of the spore wall are arranged in layers formed consecutively, beginning with the innermost layer and working outward (Tachikawa et al., 2001). The two inner layers consist primarily of mannan and 1,3-
-glucan and are similar to the vegetative cell wall (Kreger-Van Rij, 1978
). The two outer layers of the spore wall are unique to the spore and confer on the spore much of its resistance to environmental damage (Smits et al., 2001
). Outside of the
-glucan layer is a layer of chitosan, a 1,4-
-glucosamine polymer (Briza et al., 1988
). This layer is formed by the combined action of the CHS3-encoded chitin synthase and the chitin deacetylases encoded by CDA1 and CDA2 (Christodoulidou et al., 1996
; Mishra et al., 1997
; Pammer et al., 1992
). Outside of the chitosan is a thin layer that consists predominantly of cross-linked molecules of dityrosine (Briza et al., 1986
, 1990
, 1996
). The dityrosine monomers are produced in the spore cytosol by the combined action of the Dit1p and Dit2p enzymes and then exported for assembly into the wall (Briza et al., 1994
; Felder et al., 2002
). After spores are fully formed, the remains of the mother cell then collapse around the four completed spores to form the ascus. As a result, the four spores of a tetrad are enclosed together inside an ascal membrane and ascal wall, which are derived from the mother cell plasma membrane and cell wall, respectively.
In an effort to develop new assays of spore wall assembly, we examined wild-type and mutant spores by scanning electron microscopy (SEM). This analysis revealed that the wild-type spore wall has a distinctive surface texture that is altered as the dityrosine and chitosan layers are removed. Moreover, this analysis has led to the identification of a previously undescribed feature of the spore wall, the interspore bridge. These bridges connect the outer spore wall layers of adjacent spores and serve to maintain the physical association of spores upon release from the ascus.
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METHODS |
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Scanning electron microscopy.
For SEM analysis of spores, spheroplasts were first sporulated, washed once in 0·5 % SDS to remove the ascal membrane, washed once in dH2O, and then adhered to polylysine coated glass cover slips. The cells on the cover slips were fixed in 3 % glutaraldehyde in cacodylate buffer (0·1 M sodium cacodylate pH 7·4, 5 mM CaCl2) for 1 h at 23 °C, then stained for 1 h at 23 °C in 1 % osmium tetroxide and 1 % potassium ferricyanide in cacodylate buffer, washed with dH2O and then dehydrated by 10 min incubations in a graded acetone series: two incubations each in 30 %, 50 %, 70 % and 95 % acetone, and four incubations in 100 % acetone. The cover slips were then critical-point dried and sputter-coated with 4 nm of gold particles. Germinating cells, vegetative cells and asci were adhered to cover slips and prepared in the same way. Images were collected in a LEO1550 scanning electron microscope at 2·5 kV using an in-lens detector.
Transmission electron microscopy.
Cells were prepared for transmission electron microscopy (TEM) using an osmium-thiocarbohydrazide staining protocol (Rieder et al., 1996). Spores were prepared as for SEM, then fixed for 1 h in 3 % glutaraldehyde in cacodylate buffer, washed once in cacodylate buffer, resuspended in 1 % osmium tetroxide and 1 % potassium ferricyanide in cacodylate buffer, and incubated for 30 min at 23 °C. Cells were then washed four times in dH2O, resuspended in 1 % thiocarbohydrazide in water, and incubated for 5 min at 23 °C. Cells were again washed in dH2O, incubated in 1 % osmium tetroxide and 1 % potassium ferricyanide in cacodylate buffer for an additional 5 min, and washed again in dH2O. The cells were then incubated in saturated uranyl acetate for 2 h and dehydrated through a graded series of acetone washes as for the SEM samples. The dehydrated samples were embedded in Epon 812, sectioned, and images were collected on a JEOL 1200EX microscope at 80 kV.
Spore dispersal assay.
Wild-type, dit1 and chs3 mutant cells were sporulated. Zymolyase was then added to a final concentration of 0·1 mg ml1 and the spores were incubated for 15 min at 37 °C. Spores were washed once in dH2O, resuspended in dH2O, and mounted on slides for examination by phase-contrast microscopy.
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RESULTS |
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Finally, it was possible that the appearance of the spores would be altered by the treatment necessary for removal of the ascus. In particular, removal of spores from the ascus requires digestion of the ascal wall with glucanases to release the spores. Although spores are resistant to glucanase digestion, it is nonetheless possible that the enzymes could cause changes in the spore wall. To avoid this problem, vegetative cells were spheroplasted before transfer to osmotically stabilized sporulation medium. After sporulation, the spores could then be removed from the ascus without enzymic digestion. When prepared in this way, spores appeared indistinguishable by SEM from spores isolated by direct digestion of asci with glucanases (data not shown). Thus, although we cannot exclude the possibility that the surface of the spore wall is affected by preparation for electron microscopy, the appearance of the wall is not an artefact of exposure to lytic enzymes.
Spores are connected to each other by bridges
The most striking finding of the SEM analysis was the presence of connections between spores (arrowheads, Fig. 1c). To explore the nature of these interspore bridges, spores were released from asci as for the SEM analysis and examined by TEM. Interspore bridges were readily visible in these preparations as darkly staining material connecting the outer layers of the walls of adjacent spores. In these preparations, the dityrosine and chitosan layers of the spore wall stain as a single dark layer (arrowheads, Fig. 2
). This layer branches at the position of the bridges so that it both surrounds individual spores and connects adjacent spores. Thus, the bridges themselves appear to consist of chitosan and/or dityrosine.
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DISCUSSION |
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More strikingly, this analysis revealed that S. cerevisiae spores isolated from the ascus are connected by structures we have termed interspore bridges. TEM analysis of isolated spores suggested that the bridges are formed by a branching of the outer layers of the spore wall so that these layers both surround individual spores and connect adjacent spores. This inference was confirmed by analysis of chs3 mutant cells. In chs3 mutants, the interspore bridges were absent, indicating that chitin or chitosan is required for bridge construction. These results demonstrate that chitin or chitosan is essential for construction of the bridges. Whether there are additional constituents of the bridges is unclear. For instance, the bridges appear somewhat less robust in a dit1 mutants (Fig. 3) suggesting that, although bridges are still functional in dit1 mutants (Fig. 5
), dityrosine may normally be present in the bridges. Additionally, the composition of the grey-staining material in the central region of the bridges is unknown.
The existence of these bridges raises several interesting issues. For example, how are the bridges constructed? One possibility is that bridges may be formed at points of contact during spore wall construction. Perhaps contact triggers branching and interconnection of the chitosan layers of adjacent spores. Understanding bridge assembly will require the identification of the enzymes responsible for this process. It may be possible to use the spore dispersal assay (Fig. 5) to identify genes involved in bridge synthesis.
Another important question raised by the presence of these bridges is what functional role these structures play in the yeast life cycle. One possibility is suggested by the homothallic nature of S. cerevisiae. Outside of the laboratory, S. cerevisiae is found primarily in a diploid (or higher ploidy) state (Mortimer & Hawthorne, 1969). Starvation induces sporulation and the generation of haploids, but HO-mediated mating-type switching guarantees that isolated haploid spores will be able to mate and return to diploidy upon germination (Herskowitz & Jensen, 1991
). However, if haploid spores self-mate to restore diploidy, then any heterozygosities that may have been present in the parental population will be lost. A large body of literature suggests that maintenance of heterozygosity can be selectively advantageous to a population (Milton, 1997
). Therefore, one possible role for bridges is to promote, by physically connecting spores, the mating of sister spores to produce diploids and thereby restore the heterozygous state present in the parental cell. The fact that mating-type switching does not begin until the second division after germination (Strathern & Herskowitz, 1979
) might, in combination with the bridges, help promote mating between sister spores.
While this is a speculative hypothesis, some indirect support for it can be found by comparison to Schiz. pombe. Schiz. pombe is also a homothallic yeast, but unlike S. cerevisiae it exists primarily as a haploid, only mating to form diploids immediately prior to sporulation (Mortimer, 1969). Because Schiz. pombe haploids do not mate upon germination, our model would predict no role for interspore bridges in this yeast. Indeed, in Schiz. pombe, no bridges are evident by SEM and wild-type spores disperse upon release from the ascus (Gutz et al., 1974
; Nakamura et al., 2004
). It will be of interest to determine whether, in other homothallic yeasts, the presence of bridges between ascospores correlates with the tendency to mate immediately upon germination.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Briza, P., Ellinger, A., Winkler, G. & Breitenbach, M. (1988). Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan. J Biol Chem 263, 1156911574.
Briza, P., Ellinger, A., Winkler, G. & Breitenbach, M. (1990). Characterization of a DL-dityrosine-containing macromolecule from yeast ascospore walls. J Biol Chem 265, 1511815123.
Briza, P., Eckerstorfer, M. & Breitenbach, M. (1994). The sporulation-specific enzymes encoded by the DIT1 and DIT2 genes catalyze a two-step reaction leading to a soluble LL-dityrosine-containing precursor of the yeast spore wall. Proc Natl Acad Sci U S A 91, 45244528.[Abstract]
Briza, P., Kalchhauser, H., Pittenauer, E., Allmaier, G. & Breitenbach, M. (1996). N,N'-Bisformyl dityrosine is an in vivo precursor of the yeast ascospore wall. Eur J Biochem 239, 124131.[Abstract]
Christodoulidou, A., Bouriotis, V. & Thireos, G. (1996). Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae. J Biol Chem 271, 3142031425.
Felder, T., Bogengruber, E., Tenreiro, S., Ellinger, A., Sa-Correia, I. & Briza, P. (2002). Dtrlp, a multidrug resistance transporter of the major facilitator superfamily, plays an essential role in spore wall maturation in Saccharomyces cerevisiae. Eukaryot Cell 1, 799810.
Gutz, H., Heslot, H., Leupold, U. & Loprieno, N. (1974). Schizosaccharomyces pombe. In Handbook of Genetics, pp. 365446. Edited by R. C. King. New York: Plenum.
Herskowitz, I. & Jensen, R. E. (1991). Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol 194, 132146.[Medline]
Kreger-Van Rij, N. J. (1978). Electron microscopy of germinating ascospores of Saccharomyces cerevisiae. Arch Microbiol 117, 7377.[Medline]
Kupiec, M., Byers, B., Esposito, R. E. & Mitchell, A. P. (1997). Meiosis and sporulation in Saccharomyces cerevisiae. In The Molecular Biology of the Yeast Saccharomyces. Cell Cycle and Biology, pp. 8891036. Edited by J. R. Pringle, J. R. Broach & E. W. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Longtine, M. S., McKenzie, A. 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P. & Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953961.[CrossRef][Medline]
Lynn, R. R. & Magee, P. T. (1970). Development of the spore wall during ascospore formation in Saccharomyces cerevisiae. J Cell Biol 44, 688692.
Milton, J. B. (1997). Selection in Natural Populations. Oxford: Oxford University Press.
Mishra, C., Semino, C. E., McCreath, K. J., de la Vega, H., Jones, B. J., Specht, C. A. & Robbins, P. W. (1997). Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae. Yeast 13, 327336.[CrossRef][Medline]
Moens, P. B. (1971). Fine structure of ascospore development in the yeast Saccharomyces cerevisiae. Can J Microbiol 17, 507510.[Medline]
Mortimer, R. K. & Hawthorne, D. C. (1969). Yeast genetics. In The Yeasts, pp. 386453. Edited by A. H. Rose & J. S. Harrison. London: Academic Press.
Nakamura, T., Abe, H., Hirata, A. & Shimoda, C. (2004). ADAM family protein Mde10 is essential for development of spore envelopes in the fission yeast Schizosaccharomyces pombe. Eukaryot Cell 3, 2739.
Neiman, A. M. (1998). Prospore membrane formation defines a developmentally regulated branch of the secretory pathway in yeast. J Cell Biol 140, 2937.
Neiman, A. M., Katz, L. & Brennwald, P. J. (2000). Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae. Genetics 155, 16431655.
Orlean, P. (1997). Biogenesis of yeast wall and surface components. In The Molecular and Cellular Biology of the Yeast Saccharomyces. Cell Cycle and Biology, pp. 229262. Edited by J. R. Pringle, J. R. Broach & E. W. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Pammer, M., Briza, P., Ellinger, A., Schuster, T., Stucka, R., Feldmann, H. & Breitenbach, M. (1992). DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls. Yeast 8, 10891099.[Medline]
Rieder, S. E., Banta, L. M., Kohrer, K., McCaffery, J. M. & Emr, S. D. (1996). Multilamellar endosome-like compartments accumulate in the yeast vps28 vacuolar protein sorting mutant. Mol Biol Cell 7, 985999.[Abstract]
Rose, M. D. & Fink, G. R. (1990). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Smits, G. J., van den Ende, H. & Klis, F. M. (2001). Differential regulation of cell wall biogenesis during growth and development in yeast. Microbiology 147, 781794.[Medline]
Strathern, J. N. & Herskowitz, I. (1979). Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast. Cell 17, 371381.[Medline]
Tachikawa, H., Bloecher, A., Tatchell, K. & Neiman, A. M. (2001). A Gip1p-Glc7p phosphatase complex regulates septin organization and spore wall formation. J Cell Biol 155, 797808.
Received 16 April 2004;
revised 21 May 2004;
accepted 25 May 2004.
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