Limits...
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.

Show MeSH

Related in: MedlinePlus

Mapping the three LAT-A resistant alleles on the actin molecular structure. Backbone of the actin monomer from the coordinates of rabbit muscle actin as determined by Kabsch et al. (1990). Subdomains are marked I to IV. The side chains of the residues mutated to alanine in the LAT-A resistant mutants are shown and are color coded by their allele designation (Wertman et al., 1992). act1112 (yellow), act1-113 (red), act1-117 (green). The adenine nucleotide (cyan) is shown in the prominent cleft as a ball and stick model,  and the divalent cation (purple) as a Van der Waal's sphere.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139767&req=5

Figure 3: Mapping the three LAT-A resistant alleles on the actin molecular structure. Backbone of the actin monomer from the coordinates of rabbit muscle actin as determined by Kabsch et al. (1990). Subdomains are marked I to IV. The side chains of the residues mutated to alanine in the LAT-A resistant mutants are shown and are color coded by their allele designation (Wertman et al., 1992). act1112 (yellow), act1-113 (red), act1-117 (green). The adenine nucleotide (cyan) is shown in the prominent cleft as a ball and stick model, and the divalent cation (purple) as a Van der Waal's sphere.

Mentions: Thus, the halo assay results were highly indicative of a specific interaction of LAT-A with actin in yeast cells. To investigate the possibility that resistance was due to disruption of the normal LAT-A binding site on the actin molecule, we looked at the position of the three resistant alleles within the actin crystal structure. As shown in Fig. 3, the alleles define a small patch on actin adjacent to the nucleotide binding cleft. Residues which have been mutated in the three alleles (act1-112 = K213A, E214A, K215A; act1-113 = R210A, D211A; act1-117 = R183A, D184A) are thought to either directly contact through salt bridges or hydrogen bonds, the nucleotide itself, or, to form salt bridges to position other residues which themselves bind the nucleotide (Kabsch et al., 1990). We therefore determined the effect of LAT-A on nucleotide exchange.


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)

Mapping the three LAT-A resistant alleles on the actin molecular structure. Backbone of the actin monomer from the coordinates of rabbit muscle actin as determined by Kabsch et al. (1990). Subdomains are marked I to IV. The side chains of the residues mutated to alanine in the LAT-A resistant mutants are shown and are color coded by their allele designation (Wertman et al., 1992). act1112 (yellow), act1-113 (red), act1-117 (green). The adenine nucleotide (cyan) is shown in the prominent cleft as a ball and stick model,  and the divalent cation (purple) as a Van der Waal's sphere.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Mapping the three LAT-A resistant alleles on the actin molecular structure. Backbone of the actin monomer from the coordinates of rabbit muscle actin as determined by Kabsch et al. (1990). Subdomains are marked I to IV. The side chains of the residues mutated to alanine in the LAT-A resistant mutants are shown and are color coded by their allele designation (Wertman et al., 1992). act1112 (yellow), act1-113 (red), act1-117 (green). The adenine nucleotide (cyan) is shown in the prominent cleft as a ball and stick model, and the divalent cation (purple) as a Van der Waal's sphere.
Mentions: Thus, the halo assay results were highly indicative of a specific interaction of LAT-A with actin in yeast cells. To investigate the possibility that resistance was due to disruption of the normal LAT-A binding site on the actin molecule, we looked at the position of the three resistant alleles within the actin crystal structure. As shown in Fig. 3, the alleles define a small patch on actin adjacent to the nucleotide binding cleft. Residues which have been mutated in the three alleles (act1-112 = K213A, E214A, K215A; act1-113 = R210A, D211A; act1-117 = R183A, D184A) are thought to either directly contact through salt bridges or hydrogen bonds, the nucleotide itself, or, to form salt bridges to position other residues which themselves bind the nucleotide (Kabsch et al., 1990). We therefore determined the effect of LAT-A on nucleotide exchange.

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