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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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Cell cycle specificity of the effect of F-actin perturbation on SPB migration. Synchronized G1 cells of the cdc31-1 strain (JMY6-10) were isolated as in Fig. 5, and incubated at 37°C in the presence of nocodazole, together with α-factor for 2 h. The resulting shmoo-shaped cells were harvested by centrifugation and resuspended in fresh YEPD (prewarmed to 37°C) with nocodazole, but lacking α-factor, releasing them from G1 arrest and allowing bud formation. These cells were harvested after a further 1 h (small-budded cells, A) or 2 h (large-budded cells, B) and separate aliquots were treated with either 100 μM Lat-A or DMSO (controls). After another 30 min at 37°C, the cells were fixed and processed to visualize F-actin and tubulin. The position of the SPB was scored as described in Materials and Methods (n = 200 for each sample). Mothers and buds in this experiment were distinguished by shmoo-shaped (mother) or round (bud) morphology. Representative pictures of cells are shown, overlaying tubulin staining and DIC images of the same cells. Bar, 5 μm.
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Figure 6: Cell cycle specificity of the effect of F-actin perturbation on SPB migration. Synchronized G1 cells of the cdc31-1 strain (JMY6-10) were isolated as in Fig. 5, and incubated at 37°C in the presence of nocodazole, together with α-factor for 2 h. The resulting shmoo-shaped cells were harvested by centrifugation and resuspended in fresh YEPD (prewarmed to 37°C) with nocodazole, but lacking α-factor, releasing them from G1 arrest and allowing bud formation. These cells were harvested after a further 1 h (small-budded cells, A) or 2 h (large-budded cells, B) and separate aliquots were treated with either 100 μM Lat-A or DMSO (controls). After another 30 min at 37°C, the cells were fixed and processed to visualize F-actin and tubulin. The position of the SPB was scored as described in Materials and Methods (n = 200 for each sample). Mothers and buds in this experiment were distinguished by shmoo-shaped (mother) or round (bud) morphology. Representative pictures of cells are shown, overlaying tubulin staining and DIC images of the same cells. Bar, 5 μm.

Mentions: SPB position was inferred from the tubulin staining on the assumption that the SPB is at the focus of the cytoplasmic microtubule asters: only cells where this focus was obvious were scored. SPB position was classified into one of three categories: neck, indicating a distance of <0.5 μm from the mother-bud neck; mother, or bud, indicating a distance of >0.5 μm from the mother-bud neck on either side. In the experiment of Fig. 5, the bud category is expanded to include all cells with the SPB on the bud side of the neck. Mothers were distinguished from buds by morphological criteria: in Fig. 5, mothers were larger and had a distinct vacuole; in Fig. 6, mothers were shmoo-shaped; and in Fig. 9, buds were elongated. The reliability of these criteria in identifying mother and bud was tested by comparison to the actin-staining pattern in the same cells (actin patches are predominantly in the bud). In all cases, there was excellent agreement between morphological and actin-based criteria.


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)

Cell cycle specificity of the effect of F-actin perturbation on SPB migration. Synchronized G1 cells of the cdc31-1 strain (JMY6-10) were isolated as in Fig. 5, and incubated at 37°C in the presence of nocodazole, together with α-factor for 2 h. The resulting shmoo-shaped cells were harvested by centrifugation and resuspended in fresh YEPD (prewarmed to 37°C) with nocodazole, but lacking α-factor, releasing them from G1 arrest and allowing bud formation. These cells were harvested after a further 1 h (small-budded cells, A) or 2 h (large-budded cells, B) and separate aliquots were treated with either 100 μM Lat-A or DMSO (controls). After another 30 min at 37°C, the cells were fixed and processed to visualize F-actin and tubulin. The position of the SPB was scored as described in Materials and Methods (n = 200 for each sample). Mothers and buds in this experiment were distinguished by shmoo-shaped (mother) or round (bud) morphology. Representative pictures of cells are shown, overlaying tubulin staining and DIC images of the same cells. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Cell cycle specificity of the effect of F-actin perturbation on SPB migration. Synchronized G1 cells of the cdc31-1 strain (JMY6-10) were isolated as in Fig. 5, and incubated at 37°C in the presence of nocodazole, together with α-factor for 2 h. The resulting shmoo-shaped cells were harvested by centrifugation and resuspended in fresh YEPD (prewarmed to 37°C) with nocodazole, but lacking α-factor, releasing them from G1 arrest and allowing bud formation. These cells were harvested after a further 1 h (small-budded cells, A) or 2 h (large-budded cells, B) and separate aliquots were treated with either 100 μM Lat-A or DMSO (controls). After another 30 min at 37°C, the cells were fixed and processed to visualize F-actin and tubulin. The position of the SPB was scored as described in Materials and Methods (n = 200 for each sample). Mothers and buds in this experiment were distinguished by shmoo-shaped (mother) or round (bud) morphology. Representative pictures of cells are shown, overlaying tubulin staining and DIC images of the same cells. Bar, 5 μm.
Mentions: SPB position was inferred from the tubulin staining on the assumption that the SPB is at the focus of the cytoplasmic microtubule asters: only cells where this focus was obvious were scored. SPB position was classified into one of three categories: neck, indicating a distance of <0.5 μm from the mother-bud neck; mother, or bud, indicating a distance of >0.5 μm from the mother-bud neck on either side. In the experiment of Fig. 5, the bud category is expanded to include all cells with the SPB on the bud side of the neck. Mothers were distinguished from buds by morphological criteria: in Fig. 5, mothers were larger and had a distinct vacuole; in Fig. 6, mothers were shmoo-shaped; and in Fig. 9, buds were elongated. The reliability of these criteria in identifying mother and bud was tested by comparison to the actin-staining pattern in the same cells (actin patches are predominantly in the bud). In all cases, there was excellent agreement between morphological and actin-based criteria.

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
Related in: MedlinePlus