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The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

Theesfeld CL, Irazoqui JE, Bloom K, Lew DJ - J. Cell Biol. (1999)

Bottom Line: We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle.Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage.Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

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Spindle orientation in hydroxyurea-arrested cells lacking Kar9p, Bni1p, or dynein. Isogenic wild-type (W303a) and dyn1Δ (KBY62) cells, and isogenic wild-type (YEF473), kar9Δ (KBY1010), and bni1Δ (KBY1011) cells were grown in YEPD at 30°C, synchronized by addition of 200 mM hydroxyurea (HU) for 3 h (YEF473 strains) or 4 h (W303a strains), and supplemented with 0 or 100 μM Lat-A for a further 15 min, as indicated. Cells were fixed and processed to visualize F-actin and tubulin, and spindle orientation was scored as described in Materials and Methods. n = 200 for each sample.
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Figure 7: Spindle orientation in hydroxyurea-arrested cells lacking Kar9p, Bni1p, or dynein. Isogenic wild-type (W303a) and dyn1Δ (KBY62) cells, and isogenic wild-type (YEF473), kar9Δ (KBY1010), and bni1Δ (KBY1011) cells were grown in YEPD at 30°C, synchronized by addition of 200 mM hydroxyurea (HU) for 3 h (YEF473 strains) or 4 h (W303a strains), and supplemented with 0 or 100 μM Lat-A for a further 15 min, as indicated. Cells were fixed and processed to visualize F-actin and tubulin, and spindle orientation was scored as described in Materials and Methods. n = 200 for each sample.

Mentions: Previous studies have implicated a number of proteins, including the putative microtubule capturing protein Kar9p (Miller and Rose 1998), the cortical protein Bni1p (Zahner et al. 1996; Lee et al. 1999; Miller et al. 1999; Yang et al., manuscript in preparation), and the microtubule-based motor protein dynein (heavy chain Dyn1p; Li et al. 1993) in the process of spindle orientation. To address whether these proteins were important for spindle orientation late in the cell cycle, we arrested kar9Δ, bni1Δ, or dyn1Δ mutant strains with hydroxyurea. As shown in Fig. 7, none of these mutants differed from isogenic wild-type strains in terms of their ability to orient spindles. The wild-type W303 cells were not as proficient in this regard, as the wild-type YEF473 cells, presumably reflecting strain background effects. The fact that spindle orientation did (eventually) occur in these strains suggests that, given sufficient time, both the establishment and maintenance of spindle orientation can occur in the absence of these proteins.


The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

Theesfeld CL, Irazoqui JE, Bloom K, Lew DJ - J. Cell Biol. (1999)

Spindle orientation in hydroxyurea-arrested cells lacking Kar9p, Bni1p, or dynein. Isogenic wild-type (W303a) and dyn1Δ (KBY62) cells, and isogenic wild-type (YEF473), kar9Δ (KBY1010), and bni1Δ (KBY1011) cells were grown in YEPD at 30°C, synchronized by addition of 200 mM hydroxyurea (HU) for 3 h (YEF473 strains) or 4 h (W303a strains), and supplemented with 0 or 100 μM Lat-A for a further 15 min, as indicated. Cells were fixed and processed to visualize F-actin and tubulin, and spindle orientation was scored as described in Materials and Methods. n = 200 for each sample.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169477&req=5

Figure 7: Spindle orientation in hydroxyurea-arrested cells lacking Kar9p, Bni1p, or dynein. Isogenic wild-type (W303a) and dyn1Δ (KBY62) cells, and isogenic wild-type (YEF473), kar9Δ (KBY1010), and bni1Δ (KBY1011) cells were grown in YEPD at 30°C, synchronized by addition of 200 mM hydroxyurea (HU) for 3 h (YEF473 strains) or 4 h (W303a strains), and supplemented with 0 or 100 μM Lat-A for a further 15 min, as indicated. Cells were fixed and processed to visualize F-actin and tubulin, and spindle orientation was scored as described in Materials and Methods. n = 200 for each sample.
Mentions: Previous studies have implicated a number of proteins, including the putative microtubule capturing protein Kar9p (Miller and Rose 1998), the cortical protein Bni1p (Zahner et al. 1996; Lee et al. 1999; Miller et al. 1999; Yang et al., manuscript in preparation), and the microtubule-based motor protein dynein (heavy chain Dyn1p; Li et al. 1993) in the process of spindle orientation. To address whether these proteins were important for spindle orientation late in the cell cycle, we arrested kar9Δ, bni1Δ, or dyn1Δ mutant strains with hydroxyurea. As shown in Fig. 7, none of these mutants differed from isogenic wild-type strains in terms of their ability to orient spindles. The wild-type W303 cells were not as proficient in this regard, as the wild-type YEF473 cells, presumably reflecting strain background effects. The fact that spindle orientation did (eventually) occur in these strains suggests that, given sufficient time, both the establishment and maintenance of spindle orientation can occur in the absence of these proteins.

Bottom Line: We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle.Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage.Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

Show MeSH