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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 actin polymerization. Purified actin from ACT1 or  act1-117 cells was incubated  with LAT-A before addition  of polymerization salts. (a)  After polymerization actin  was spun to pellet filamentous actin. Supernatants and  pellets were run on gels and  actin was visualized using  Coomassie staining. (b)  Quantitation of the data in a  using densitometry. (c) After  polymerization, Rd-phalloidin–labeled actin filaments  were visualized using fluorescence microscopy. Bar, 5 μM.
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Figure 5: The effect of LAT-A on actin polymerization. Purified actin from ACT1 or act1-117 cells was incubated with LAT-A before addition of polymerization salts. (a) After polymerization actin was spun to pellet filamentous actin. Supernatants and pellets were run on gels and actin was visualized using Coomassie staining. (b) Quantitation of the data in a using densitometry. (c) After polymerization, Rd-phalloidin–labeled actin filaments were visualized using fluorescence microscopy. Bar, 5 μM.

Mentions: As a final test of LAT-A specificity, we purified actin from two of the LAT-A resistant strains, act1-113 and act1-117. We did not attempt to isolate actin from act1-112 cells because they are very sick and we therefore thought the likelihood that they would yield an actin sufficiently stable to allow in vitro analysis was low. Indeed, actin from an act1-113 mutant polymerized very poorly in vitro (<10% could be pelleted under conditions that nearly quantitatively pellet polymerized wild-type actin), even though act1-113 strains are considerably more healthy than act1112 strains. Since it would have been difficult to analyze data from assembly of the act1-113 actin, we proceeded with only act1-117 actin. G-actin from ACT1 or act1-117 strains was allowed to polymerize in the absence or presence of LAT-A. F-actin was pelleted, and the resulting supernatants and pellets were analyzed by SDS-PAGE (Fig. 5 a). The actin protein bands on this gel were analyzed by densitometry (Fig. 5 b). The results from these experiments show that incubation of LAT-A with an equimolar concentration of wild-type actin, but not actin from act1117 cells, resulted in inhibition of filament assembly. The act1-117 mutation itself appears to affect the ability of actin to form filaments. Only ∼40% of act1-117 actin was pelleted using the conditions which resulted in >90% pelleting for wild-type actin. However, the addition of LAT-A to this actin did not significantly affect its ability to polymerize. To verify that pelleting of the mutant actin was due to filament formation, rather than aggregation of actin monomers, we observed the mutant actin filaments, labeled with Rh-phalloidin, directly by fluorescence microscopy (Fig. 5 c) and found that both wild-type and act1-117 actin were able to form filaments, but only act1-117 actin was able to form filaments in the presence 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)

The effect of LAT-A  on actin polymerization. Purified actin from ACT1 or  act1-117 cells was incubated  with LAT-A before addition  of polymerization salts. (a)  After polymerization actin  was spun to pellet filamentous actin. Supernatants and  pellets were run on gels and  actin was visualized using  Coomassie staining. (b)  Quantitation of the data in a  using densitometry. (c) After  polymerization, Rd-phalloidin–labeled actin filaments  were visualized using fluorescence microscopy. Bar, 5 μM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: The effect of LAT-A on actin polymerization. Purified actin from ACT1 or act1-117 cells was incubated with LAT-A before addition of polymerization salts. (a) After polymerization actin was spun to pellet filamentous actin. Supernatants and pellets were run on gels and actin was visualized using Coomassie staining. (b) Quantitation of the data in a using densitometry. (c) After polymerization, Rd-phalloidin–labeled actin filaments were visualized using fluorescence microscopy. Bar, 5 μM.
Mentions: As a final test of LAT-A specificity, we purified actin from two of the LAT-A resistant strains, act1-113 and act1-117. We did not attempt to isolate actin from act1-112 cells because they are very sick and we therefore thought the likelihood that they would yield an actin sufficiently stable to allow in vitro analysis was low. Indeed, actin from an act1-113 mutant polymerized very poorly in vitro (<10% could be pelleted under conditions that nearly quantitatively pellet polymerized wild-type actin), even though act1-113 strains are considerably more healthy than act1112 strains. Since it would have been difficult to analyze data from assembly of the act1-113 actin, we proceeded with only act1-117 actin. G-actin from ACT1 or act1-117 strains was allowed to polymerize in the absence or presence of LAT-A. F-actin was pelleted, and the resulting supernatants and pellets were analyzed by SDS-PAGE (Fig. 5 a). The actin protein bands on this gel were analyzed by densitometry (Fig. 5 b). The results from these experiments show that incubation of LAT-A with an equimolar concentration of wild-type actin, but not actin from act1117 cells, resulted in inhibition of filament assembly. The act1-117 mutation itself appears to affect the ability of actin to form filaments. Only ∼40% of act1-117 actin was pelleted using the conditions which resulted in >90% pelleting for wild-type actin. However, the addition of LAT-A to this actin did not significantly affect its ability to polymerize. To verify that pelleting of the mutant actin was due to filament formation, rather than aggregation of actin monomers, we observed the mutant actin filaments, labeled with Rh-phalloidin, directly by fluorescence microscopy (Fig. 5 c) and found that both wild-type and act1-117 actin were able to form filaments, but only act1-117 actin was able to form filaments in the presence 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