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High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A.

Ayscough KR, Stryker J, Pokala N, Sanders M, Crews P, Drubin DG - J. Cell Biol. (1997)

Bottom Line: Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability.Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.Thus, actin filaments are also required for maintenance of an axis of cell polarity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202, USA.

ABSTRACT
We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

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Sensitivity of a set of congenic actin mutants to LAT-A.  Halo assays were used to assess the sensitivity of 23 actin mutants  to LAT-A. (a–d) Representative examples of LAT-A halo assays. Concentrations measured 0.5 mM, 1 mM, and 2 mM. (a)  ACT1 (wild-type actin), (b) act1-117, (c) act1-129, (d) act1-116.  (e) Summary bar graph of the relative apparent sensitivities of all  of the actin alleles tested compared to the wild-type.
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Figure 2: Sensitivity of a set of congenic actin mutants to LAT-A. Halo assays were used to assess the sensitivity of 23 actin mutants to LAT-A. (a–d) Representative examples of LAT-A halo assays. Concentrations measured 0.5 mM, 1 mM, and 2 mM. (a) ACT1 (wild-type actin), (b) act1-117, (c) act1-129, (d) act1-116. (e) Summary bar graph of the relative apparent sensitivities of all of the actin alleles tested compared to the wild-type.

Mentions: As LAT-A provided an opportunity to determine for the first time the consequences of a total lack of filamentous actin in yeast, it was necessary to first demonstrate the specificity of the drug in living yeast cells. To demonstrate LAT-A specificity, we took advantage of a congenic collection of actin mutants which was generated by a charged-to-alanine mutagenesis scan of ACT1, the single, yeast conventional actin gene (Wertman et al., 1992). Each allele in the collection expresses a mutant form of actin as the sole source of actin in the cell. The collection, comprising 23 viable strains, was previously used to map the binding sites on the actin surface for phalloidin (Drubin et al., 1993), for yeast fimbrin (Holtzman et al., 1994), and for several other actin-associated proteins (Amberg et al., 1995). The LAT-A sensitivities of all of the strains in the collection were compared using the halo assay. Fig. 2, a–d shows the halo assays of several strains in the collection to illustrate the variation in sensitivity among the alleles. In addition, the LAT-A sensitivity of each allele was compared to the sensitivity of cells containing wild-type actin. This comparison is shown graphically in Fig. 2 e.


High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A.

Ayscough KR, Stryker J, Pokala N, Sanders M, Crews P, Drubin DG - J. Cell Biol. (1997)

Sensitivity of a set of congenic actin mutants to LAT-A.  Halo assays were used to assess the sensitivity of 23 actin mutants  to LAT-A. (a–d) Representative examples of LAT-A halo assays. Concentrations measured 0.5 mM, 1 mM, and 2 mM. (a)  ACT1 (wild-type actin), (b) act1-117, (c) act1-129, (d) act1-116.  (e) Summary bar graph of the relative apparent sensitivities of all  of the actin alleles tested compared to the wild-type.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139767&req=5

Figure 2: Sensitivity of a set of congenic actin mutants to LAT-A. Halo assays were used to assess the sensitivity of 23 actin mutants to LAT-A. (a–d) Representative examples of LAT-A halo assays. Concentrations measured 0.5 mM, 1 mM, and 2 mM. (a) ACT1 (wild-type actin), (b) act1-117, (c) act1-129, (d) act1-116. (e) Summary bar graph of the relative apparent sensitivities of all of the actin alleles tested compared to the wild-type.
Mentions: As LAT-A provided an opportunity to determine for the first time the consequences of a total lack of filamentous actin in yeast, it was necessary to first demonstrate the specificity of the drug in living yeast cells. To demonstrate LAT-A specificity, we took advantage of a congenic collection of actin mutants which was generated by a charged-to-alanine mutagenesis scan of ACT1, the single, yeast conventional actin gene (Wertman et al., 1992). Each allele in the collection expresses a mutant form of actin as the sole source of actin in the cell. The collection, comprising 23 viable strains, was previously used to map the binding sites on the actin surface for phalloidin (Drubin et al., 1993), for yeast fimbrin (Holtzman et al., 1994), and for several other actin-associated proteins (Amberg et al., 1995). The LAT-A sensitivities of all of the strains in the collection were compared using the halo assay. Fig. 2, a–d shows the halo assays of several strains in the collection to illustrate the variation in sensitivity among the alleles. In addition, the LAT-A sensitivity of each allele was compared to the sensitivity of cells containing wild-type actin. This comparison is shown graphically in Fig. 2 e.

Bottom Line: Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability.Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.Thus, actin filaments are also required for maintenance of an axis of cell polarity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202, USA.

ABSTRACT
We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

Show MeSH
Related in: MedlinePlus