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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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Immunolocalization of Spa2p in cells exiting stationary phase in the absence or presence of LAT-A. (a) Quantification of immunofluorescence to assess the percentage of cells that  exhibited polarized Spa2p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of  Spa2p in cells grown in the absence (b) or presence (c) of LAT-A.  Bar, 5 μm.
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Figure 12: Immunolocalization of Spa2p in cells exiting stationary phase in the absence or presence of LAT-A. (a) Quantification of immunofluorescence to assess the percentage of cells that exhibited polarized Spa2p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of Spa2p in cells grown in the absence (b) or presence (c) of LAT-A. Bar, 5 μm.

Mentions: Spa2p localizes to the presumptive bud site and to the tip of mating projections (Snyder, 1989; Snyder et al., 1991). Cells deleted for SPA2 exhibit no defects in the ability to bud but show a defect in bud site selection (Snyder, 1989). In the absence of LAT-A, Spa2p became polarized with similar kinetics to actin (Fig. 12, a and b). In the presence of LAT-A, Spa2p was still able to polarize. However, the kinetics of polarization were delayed (Fig. 12, a and c). In three separate experiments, polarized localization of Spa2p was always 1–2 h slower than in the absence of LAT-A.


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)

Immunolocalization of Spa2p in cells exiting stationary phase in the absence or presence of LAT-A. (a) Quantification of immunofluorescence to assess the percentage of cells that  exhibited polarized Spa2p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of  Spa2p in cells grown in the absence (b) or presence (c) of LAT-A.  Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Immunolocalization of Spa2p in cells exiting stationary phase in the absence or presence of LAT-A. (a) Quantification of immunofluorescence to assess the percentage of cells that exhibited polarized Spa2p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of Spa2p in cells grown in the absence (b) or presence (c) of LAT-A. Bar, 5 μm.
Mentions: Spa2p localizes to the presumptive bud site and to the tip of mating projections (Snyder, 1989; Snyder et al., 1991). Cells deleted for SPA2 exhibit no defects in the ability to bud but show a defect in bud site selection (Snyder, 1989). In the absence of LAT-A, Spa2p became polarized with similar kinetics to actin (Fig. 12, a and b). In the presence of LAT-A, Spa2p was still able to polarize. However, the kinetics of polarization were delayed (Fig. 12, a and c). In three separate experiments, polarized localization of Spa2p was always 1–2 h slower than in the absence of LAT-A.

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