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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 formation of two-budded cells after incubation of  wild-type cells with LAT-A. Cells were released from stationary  phase and allowed to grow until more than 50% of the population was small budded. Cells were then treated with LAT-A for 5  min (or with an equal volume of DMSO in the control case).  They were then washed to remove LAT-A and allowed to resume growth. After 2 1/2 h the morphology of cells was assessed.  Examples of two-budded haploid cells (a) and diploid cells (c)  are shown. Cells were classified as unbudded, single-budded, or  two-budded in the haploid (b) or diploid (d) populations. Control  cells (▪); cells treated with LAT-A (▨ ).
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Figure 14: The formation of two-budded cells after incubation of wild-type cells with LAT-A. Cells were released from stationary phase and allowed to grow until more than 50% of the population was small budded. Cells were then treated with LAT-A for 5 min (or with an equal volume of DMSO in the control case). They were then washed to remove LAT-A and allowed to resume growth. After 2 1/2 h the morphology of cells was assessed. Examples of two-budded haploid cells (a) and diploid cells (c) are shown. Cells were classified as unbudded, single-budded, or two-budded in the haploid (b) or diploid (d) populations. Control cells (▪); cells treated with LAT-A (▨ ).

Mentions: To determine whether actin plays a role in the maintenance of the axis of cell polarity, we released both haploid and diploid cells from stationary phase to a stage where more than 50% of cells had small buds. We then treated these cells with LAT-A for 5 min to completely disrupt the actin cytoskeleton, washed the cells, and allowed them to resume growth. After 2 h, we analyzed the morphology of the cells (Fig. 14). For both haploid and diploid cells, the disruption of the actin cytoskeleton caused a significant percentage of cells to become two-budded. Interestingly, in nearly all cases, this second bud was correctly placed with respect to the expected position of bud site selection. That is, haploid buds were adjacent to one another, and diploid buds were either at both poles or at the same pole of the cell (for a discussion of budding patterns, see Drubin and Nelson, 1996).


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 formation of two-budded cells after incubation of  wild-type cells with LAT-A. Cells were released from stationary  phase and allowed to grow until more than 50% of the population was small budded. Cells were then treated with LAT-A for 5  min (or with an equal volume of DMSO in the control case).  They were then washed to remove LAT-A and allowed to resume growth. After 2 1/2 h the morphology of cells was assessed.  Examples of two-budded haploid cells (a) and diploid cells (c)  are shown. Cells were classified as unbudded, single-budded, or  two-budded in the haploid (b) or diploid (d) populations. Control  cells (▪); cells treated with LAT-A (▨ ).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 14: The formation of two-budded cells after incubation of wild-type cells with LAT-A. Cells were released from stationary phase and allowed to grow until more than 50% of the population was small budded. Cells were then treated with LAT-A for 5 min (or with an equal volume of DMSO in the control case). They were then washed to remove LAT-A and allowed to resume growth. After 2 1/2 h the morphology of cells was assessed. Examples of two-budded haploid cells (a) and diploid cells (c) are shown. Cells were classified as unbudded, single-budded, or two-budded in the haploid (b) or diploid (d) populations. Control cells (▪); cells treated with LAT-A (▨ ).
Mentions: To determine whether actin plays a role in the maintenance of the axis of cell polarity, we released both haploid and diploid cells from stationary phase to a stage where more than 50% of cells had small buds. We then treated these cells with LAT-A for 5 min to completely disrupt the actin cytoskeleton, washed the cells, and allowed them to resume growth. After 2 h, we analyzed the morphology of the cells (Fig. 14). For both haploid and diploid cells, the disruption of the actin cytoskeleton caused a significant percentage of cells to become two-budded. Interestingly, in nearly all cases, this second bud was correctly placed with respect to the expected position of bud site selection. That is, haploid buds were adjacent to one another, and diploid buds were either at both poles or at the same pole of the cell (for a discussion of budding patterns, see Drubin and Nelson, 1996).

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