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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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The effect of LAT-A on growth and viability of cells. Growth of wild-type cells in the  presence (•) or absence (○) of LAT-A assessed  by cell counting (a) or by OD measurements (b).  Growth of mutant cells in the presence (•) or  absence (○) of LAT-A assessed by cell counting,  (c) act1-117, and (d) act1-113. (e) Viability of  wild-type cells following addition of 100 μM  LAT-A to a log phase culture.
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Figure 6: The effect of LAT-A on growth and viability of cells. Growth of wild-type cells in the presence (•) or absence (○) of LAT-A assessed by cell counting (a) or by OD measurements (b). Growth of mutant cells in the presence (•) or absence (○) of LAT-A assessed by cell counting, (c) act1-117, and (d) act1-113. (e) Viability of wild-type cells following addition of 100 μM LAT-A to a log phase culture.

Mentions: Having determined that the effects of LAT-A on actin in yeast cells are rapid, reversible, and specific, we next determined how total disruption of actin filaments would affect cellular processes. The growth of cells in rich medium was monitored by cell counting following the addition of LAT-A (Fig. 6 a). After addition of the drug there was almost no increase in cell number. However, when cell growth was assessed by measuring turbidity (OD 600 nm), there was an increase after LAT-A addition to about 2.5 times the original OD reading (Fig. 6 b). This indicated a growth in cell volume that was not matched by increasing cell number. The mutant alleles act1-113 and act1-117 were also assessed for growth in the presence of LAT-A. Neither strain showed detectable alteration of its growth rate in the presence of the drug (Fig. 6, c and d). In addition, both act1-113 and act1-117 mutants were stained for F-actin in the presence of LAT-A. Cortical actin patches were clearly present in these cells throughout the 1-h time course (data not shown).


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)

The effect of LAT-A on growth and viability of cells. Growth of wild-type cells in the  presence (•) or absence (○) of LAT-A assessed  by cell counting (a) or by OD measurements (b).  Growth of mutant cells in the presence (•) or  absence (○) of LAT-A assessed by cell counting,  (c) act1-117, and (d) act1-113. (e) Viability of  wild-type cells following addition of 100 μM  LAT-A to a log phase culture.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: The effect of LAT-A on growth and viability of cells. Growth of wild-type cells in the presence (•) or absence (○) of LAT-A assessed by cell counting (a) or by OD measurements (b). Growth of mutant cells in the presence (•) or absence (○) of LAT-A assessed by cell counting, (c) act1-117, and (d) act1-113. (e) Viability of wild-type cells following addition of 100 μM LAT-A to a log phase culture.
Mentions: Having determined that the effects of LAT-A on actin in yeast cells are rapid, reversible, and specific, we next determined how total disruption of actin filaments would affect cellular processes. The growth of cells in rich medium was monitored by cell counting following the addition of LAT-A (Fig. 6 a). After addition of the drug there was almost no increase in cell number. However, when cell growth was assessed by measuring turbidity (OD 600 nm), there was an increase after LAT-A addition to about 2.5 times the original OD reading (Fig. 6 b). This indicated a growth in cell volume that was not matched by increasing cell number. The mutant alleles act1-113 and act1-117 were also assessed for growth in the presence of LAT-A. Neither strain showed detectable alteration of its growth rate in the presence of the drug (Fig. 6, c and d). In addition, both act1-113 and act1-117 mutants were stained for F-actin in the presence of LAT-A. Cortical actin patches were clearly present in these cells throughout the 1-h time course (data not shown).

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