Abstract
We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton- plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.
Full Text
The Full Text of this article is available as a PDF (5.0 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adams A. E., Botstein D., Drubin D. G. Requirement of yeast fimbrin for actin organization and morphogenesis in vivo. Nature. 1991 Dec 5;354(6352):404–408. doi: 10.1038/354404a0. [DOI] [PubMed] [Google Scholar]
- Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barnes G., Drubin D. G., Stearns T. The cytoskeleton of Saccharomyces cerevisiae. Curr Opin Cell Biol. 1990 Feb;2(1):109–115. doi: 10.1016/s0955-0674(05)80040-7. [DOI] [PubMed] [Google Scholar]
- Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
- Chowdhury S., Smith K. W., Gustin M. C. Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation. J Cell Biol. 1992 Aug;118(3):561–571. doi: 10.1083/jcb.118.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cosgrove D. Biophysical control of plant cell growth. Annu Rev Plant Physiol. 1986;37:377–405. doi: 10.1146/annurev.pp.37.060186.002113. [DOI] [PubMed] [Google Scholar]
- Drubin D. G. Development of cell polarity in budding yeast. Cell. 1991 Jun 28;65(7):1093–1096. doi: 10.1016/0092-8674(91)90001-f. [DOI] [PubMed] [Google Scholar]
- Drubin D. G., Jones H. D., Wertman K. F. Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Mol Biol Cell. 1993 Dec;4(12):1277–1294. doi: 10.1091/mbc.4.12.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drubin D. G., Miller K. G., Botstein D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J Cell Biol. 1988 Dec;107(6 Pt 2):2551–2561. doi: 10.1083/jcb.107.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greer C., Schekman R. Calcium control of Saccharomyces cerevisiae actin assembly. Mol Cell Biol. 1982 Oct;2(10):1279–1286. doi: 10.1128/mcb.2.10.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harold F. M. To shape a cell: an inquiry into the causes of morphogenesis of microorganisms. Microbiol Rev. 1990 Dec;54(4):381–431. doi: 10.1128/mr.54.4.381-431.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koch A. L. Biophysics of bacterial walls viewed as stress-bearing fabric. Microbiol Rev. 1988 Sep;52(3):337–353. doi: 10.1128/mr.52.3.337-353.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koch A. L. How bacteria grow and divide in spite of internal hydrostatic pressure. Can J Microbiol. 1985 Dec;31(12):1071–1084. doi: 10.1139/m85-204. [DOI] [PubMed] [Google Scholar]
- Koch A. L. The surface stress theory of microbial morphogenesis. Adv Microb Physiol. 1983;24:301–366. doi: 10.1016/s0065-2911(08)60388-4. [DOI] [PubMed] [Google Scholar]
- Kübler E., Riezman H. Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J. 1993 Jul;12(7):2855–2862. doi: 10.1002/j.1460-2075.1993.tb05947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu H. P., Bretscher A. Disruption of the single tropomyosin gene in yeast results in the disappearance of actin cables from the cytoskeleton. Cell. 1989 Apr 21;57(2):233–242. doi: 10.1016/0092-8674(89)90961-6. [DOI] [PubMed] [Google Scholar]
- Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
- Moon A. L., Janmey P. A., Louie K. A., Drubin D. G. Cofilin is an essential component of the yeast cortical cytoskeleton. J Cell Biol. 1993 Jan;120(2):421–435. doi: 10.1083/jcb.120.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
- Ortega J. K., Zehr E. G., Keanini R. G. In vivo creep and stress relaxation experiments to determine the wall extensibility and yield threshold for the sporangiophores of phycomyces. Biophys J. 1989 Sep;56(3):465–475. doi: 10.1016/S0006-3495(89)82694-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Preuss D., Mulholland J., Franzusoff A., Segev N., Botstein D. Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol Biol Cell. 1992 Jul;3(7):789–803. doi: 10.1091/mbc.3.7.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Preuss D., Mulholland J., Kaiser C. A., Orlean P., Albright C., Rose M. D., Robbins P. W., Botstein D. Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy. Yeast. 1991 Dec;7(9):891–911. doi: 10.1002/yea.320070902. [DOI] [PubMed] [Google Scholar]
- Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
- REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shockman G. D., Daneo-Moore L., Higgins M. L. Problems of cell wall and membrane growth, enlargement, and division. Ann N Y Acad Sci. 1974 May 10;235(0):161–197. doi: 10.1111/j.1749-6632.1974.tb43265.x. [DOI] [PubMed] [Google Scholar]
- Wertman K. F., Drubin D. G., Botstein D. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 1992 Oct;132(2):337–350. doi: 10.1093/genetics/132.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wright R., Rine J. Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy. Methods Cell Biol. 1989;31:473–512. doi: 10.1016/s0091-679x(08)61624-6. [DOI] [PubMed] [Google Scholar]
- Yaffe M. P. Organelle inheritance in the yeast cell cycle. Trends Cell Biol. 1991 Dec;1(6):160–164. doi: 10.1016/0962-8924(91)90017-4. [DOI] [PubMed] [Google Scholar]
- Zechel K. Isolation of polymerization-competent cytoplasmic actin by affinity chromatography on immobilized DNAse I using formamide as eluant. Eur J Biochem. 1980 Sep;110(2):343–348. doi: 10.1111/j.1432-1033.1980.tb04873.x. [DOI] [PubMed] [Google Scholar]
- van Tuinen E., Riezman H. Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level. J Histochem Cytochem. 1987 Mar;35(3):327–333. doi: 10.1177/35.3.3546482. [DOI] [PubMed] [Google Scholar]