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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 calmodulin 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 calmodulin staining when incubated in  the presence (•) or absence (○) of LAT-A. Immunolocalization  of calmodulin in cells grown in the absence (b) or presence (c) of  LAT-A. Bar, 5 μm.
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Figure 10: Immunolocalization of calmodulin 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 calmodulin staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of calmodulin in cells grown in the absence (b) or presence (c) of LAT-A. Bar, 5 μm.

Mentions: We found that actin is required for the localization of Sec4p, Sec8p, and Smy1p to the presumptive bud site. In contrast, both Myo2p and calmodulin were able to localize to this region in the absence of actin, albeit to a lesser extent than in control cells. Examples of kinetic and photographic data are shown for Sec4p and calmodulin (Figs. 9 and 10). In cells growing out from G0, Sec4p became highly polarized at the presumptive bud site, remaining at the bud tip in small-budded cells and then becoming slightly more diffuse as the bud grows. It was not clearly visible in most medium- to large-budded cells but was observed at the mother-bud neck before cytokinesis (Fig. 9, a and b). However, when LAT-A was added to the growth medium during the exit from stationary phase, Sec4p did not exhibit polarized localization (Fig. 9, a and 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 calmodulin 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 calmodulin staining when incubated in  the presence (•) or absence (○) of LAT-A. Immunolocalization  of calmodulin 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 10: Immunolocalization of calmodulin 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 calmodulin staining when incubated in the presence (•) or absence (○) of LAT-A. Immunolocalization of calmodulin in cells grown in the absence (b) or presence (c) of LAT-A. Bar, 5 μm.
Mentions: We found that actin is required for the localization of Sec4p, Sec8p, and Smy1p to the presumptive bud site. In contrast, both Myo2p and calmodulin were able to localize to this region in the absence of actin, albeit to a lesser extent than in control cells. Examples of kinetic and photographic data are shown for Sec4p and calmodulin (Figs. 9 and 10). In cells growing out from G0, Sec4p became highly polarized at the presumptive bud site, remaining at the bud tip in small-budded cells and then becoming slightly more diffuse as the bud grows. It was not clearly visible in most medium- to large-budded cells but was observed at the mother-bud neck before cytokinesis (Fig. 9, a and b). However, when LAT-A was added to the growth medium during the exit from stationary phase, Sec4p did not exhibit polarized localization (Fig. 9, a and 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