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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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Model depicting  the development of polarity  at the presumptive bud site.  Cdc42p and Bem1p localize  to the presumptive bud site  in an actin-independent manner. After localization of  these polarity establishment  proteins, other proteins associated with the development  of cell polarity are able to localize. Proteins associated  with secretion require actin  in order to achieve their polarized localization. Other proteins do not require actin for  localization and therefore occupy a separate branch in polarity development. Note that  in the case of Cdc10p, Cdc11p,  and Spa2p, a pathway parallel to the actin/secretion pathway is indicated by results  from previous studies (see  text). Additional experiments  are required to elucidate dependency relationships for localization of Cdc10p, Cdc11p,  Spa2p, Bni4p, and Gin4p.
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Figure 15: Model depicting the development of polarity at the presumptive bud site. Cdc42p and Bem1p localize to the presumptive bud site in an actin-independent manner. After localization of these polarity establishment proteins, other proteins associated with the development of cell polarity are able to localize. Proteins associated with secretion require actin in order to achieve their polarized localization. Other proteins do not require actin for localization and therefore occupy a separate branch in polarity development. Note that in the case of Cdc10p, Cdc11p, and Spa2p, a pathway parallel to the actin/secretion pathway is indicated by results from previous studies (see text). Additional experiments are required to elucidate dependency relationships for localization of Cdc10p, Cdc11p, Spa2p, Bni4p, and Gin4p.

Mentions: Fig. 15 shows a hierarchy of protein functions required for cell polarity development. Previous studies suggested that Cdc42p and Bem1p function early in the pathways leading to polarity establishment. Temperature-conditional cdc42 and bem1 mutations caused the actin cytoskeleton and septins to be delocalized at the nonpermissive temperature, indicating that cytoskeleton localization depends on the functions of the polarity establishment proteins (Adams et al., 1990; Bender and Pringle, 1991; Chant et al., 1991; Pringle et al., 1995). We showed here that neither Cdc42p nor Bem1p requires actin for localization, an observation consistent with the conclusion that Cdc42p becomes localized independent of actin and then organizes the actin cytoskeleton.


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)

Model depicting  the development of polarity  at the presumptive bud site.  Cdc42p and Bem1p localize  to the presumptive bud site  in an actin-independent manner. After localization of  these polarity establishment  proteins, other proteins associated with the development  of cell polarity are able to localize. Proteins associated  with secretion require actin  in order to achieve their polarized localization. Other proteins do not require actin for  localization and therefore occupy a separate branch in polarity development. Note that  in the case of Cdc10p, Cdc11p,  and Spa2p, a pathway parallel to the actin/secretion pathway is indicated by results  from previous studies (see  text). Additional experiments  are required to elucidate dependency relationships for localization of Cdc10p, Cdc11p,  Spa2p, Bni4p, and Gin4p.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 15: Model depicting the development of polarity at the presumptive bud site. Cdc42p and Bem1p localize to the presumptive bud site in an actin-independent manner. After localization of these polarity establishment proteins, other proteins associated with the development of cell polarity are able to localize. Proteins associated with secretion require actin in order to achieve their polarized localization. Other proteins do not require actin for localization and therefore occupy a separate branch in polarity development. Note that in the case of Cdc10p, Cdc11p, and Spa2p, a pathway parallel to the actin/secretion pathway is indicated by results from previous studies (see text). Additional experiments are required to elucidate dependency relationships for localization of Cdc10p, Cdc11p, Spa2p, Bni4p, and Gin4p.
Mentions: Fig. 15 shows a hierarchy of protein functions required for cell polarity development. Previous studies suggested that Cdc42p and Bem1p function early in the pathways leading to polarity establishment. Temperature-conditional cdc42 and bem1 mutations caused the actin cytoskeleton and septins to be delocalized at the nonpermissive temperature, indicating that cytoskeleton localization depends on the functions of the polarity establishment proteins (Adams et al., 1990; Bender and Pringle, 1991; Chant et al., 1991; Pringle et al., 1995). We showed here that neither Cdc42p nor Bem1p requires actin for localization, an observation consistent with the conclusion that Cdc42p becomes localized independent of actin and then organizes the actin cytoskeleton.

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