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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 Cdc11p in cells exiting stationary phase in the absence or presence of LAT-A. (c) Quantification of immunofluorescence to assess the percentage of cells that  exhibited polarized Cdc11p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of  Cdc11p in cells grown in the absence (b) or presence (c) of LAT-A.  Note the lack of double ring structures in the cells incubated with  LAT-A. Bar, 5 μm.
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Figure 11: Immunolocalization of Cdc11p in cells exiting stationary phase in the absence or presence of LAT-A. (c) Quantification of immunofluorescence to assess the percentage of cells that exhibited polarized Cdc11p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of Cdc11p in cells grown in the absence (b) or presence (c) of LAT-A. Note the lack of double ring structures in the cells incubated with LAT-A. Bar, 5 μm.

Mentions: We examined the localization of two septin proteins, Cdc10p and Cdc11p. Because the results were indistinguishable for these two proteins, we have only presented the data for Cdc11p. In both the absence and presence of LAT-A, Cdc11p became polarized and the kinetics of localization were similar (Fig. 11). The staining pattern was a bright ring structure at the presumptive bud site. In the absence of LAT-A, cells with larger buds were seen to contain a double ring of staining at the bud neck (Fig. 11 b). In dividing cells, one ring appeared to remain with the mother and the other with the daughter cell. In the presence of LAT-A, only single rings were seen, indicating that ring duplication is dependent on bud formation (Fig. 11 c).


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 Cdc11p in cells exiting stationary phase in the absence or presence of LAT-A. (c) Quantification of immunofluorescence to assess the percentage of cells that  exhibited polarized Cdc11p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of  Cdc11p in cells grown in the absence (b) or presence (c) of LAT-A.  Note the lack of double ring structures in the cells incubated with  LAT-A. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: Immunolocalization of Cdc11p in cells exiting stationary phase in the absence or presence of LAT-A. (c) Quantification of immunofluorescence to assess the percentage of cells that exhibited polarized Cdc11p staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of Cdc11p in cells grown in the absence (b) or presence (c) of LAT-A. Note the lack of double ring structures in the cells incubated with LAT-A. Bar, 5 μm.
Mentions: We examined the localization of two septin proteins, Cdc10p and Cdc11p. Because the results were indistinguishable for these two proteins, we have only presented the data for Cdc11p. In both the absence and presence of LAT-A, Cdc11p became polarized and the kinetics of localization were similar (Fig. 11). The staining pattern was a bright ring structure at the presumptive bud site. In the absence of LAT-A, cells with larger buds were seen to contain a double ring of staining at the bud neck (Fig. 11 b). In dividing cells, one ring appeared to remain with the mother and the other with the daughter cell. In the presence of LAT-A, only single rings were seen, indicating that ring duplication is dependent on bud formation (Fig. 11 c).

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