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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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F-actin dependence of spindle orientation in synchronized cells. Wild-type (DLY1) cells were grown in YEPD at 30°C, synchronized by the addition of 25 ng/ml α-factor for 3 h, harvested, and resuspended in fresh YEPD. At 30, 45, and 60 min after release from α-factor, 6.25 μM Lat-A (or DMSO for controls) was added to aliquots of cells, which were then fixed at 75 min and processed to visualize tubulin. A, Kinetics of bud formation after release from α-factor. B, Quantification of preanaphase spindle orientation (scored as described in Materials and Methods). White bars, control; black bars, Lat-A treated. C, Quantification of postanaphase spindle orientation. White bars, control; black bars, Lat-A treated. D, Quantification of Lat-A induced errors observed in preanaphase (white bars) and postanaphase (black bars) spindles. To derive the proportion of cells displaying Lat-A–induced spindle misorientation, we subtracted the percent of misoriented spindles in control cells from that in Lat-A treated cells from the same sample. At least 100 cells were scored for each sample.
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Figure 4: F-actin dependence of spindle orientation in synchronized cells. Wild-type (DLY1) cells were grown in YEPD at 30°C, synchronized by the addition of 25 ng/ml α-factor for 3 h, harvested, and resuspended in fresh YEPD. At 30, 45, and 60 min after release from α-factor, 6.25 μM Lat-A (or DMSO for controls) was added to aliquots of cells, which were then fixed at 75 min and processed to visualize tubulin. A, Kinetics of bud formation after release from α-factor. B, Quantification of preanaphase spindle orientation (scored as described in Materials and Methods). White bars, control; black bars, Lat-A treated. C, Quantification of postanaphase spindle orientation. White bars, control; black bars, Lat-A treated. D, Quantification of Lat-A induced errors observed in preanaphase (white bars) and postanaphase (black bars) spindles. To derive the proportion of cells displaying Lat-A–induced spindle misorientation, we subtracted the percent of misoriented spindles in control cells from that in Lat-A treated cells from the same sample. At least 100 cells were scored for each sample.

Mentions: To examine whether cells traversing the cell cycle require actin to establish and/or maintain spindle orientation, a proliferating asynchronous population of wild-type cells was treated for 30 min with Lat-A. This induced a dramatic spindle misorientation in 53% of the cells (Fig. 3). Misaligned preanaphase spindles were observed in small- and medium-budded cells, but at lower frequency in large-budded cells (see below, Fig. 4). In addition, postanaphase spindles in medium-budded cells were often misaligned, probably resulting from elongation of short spindles that had become misoriented soon after Lat-A addition (Fig. 3 A; we return to this point in the Discussion). Thus, spindle orientation is actin-dependent in some fraction of a proliferating cell population, but not in hydroxyurea-arrested cells.


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)

F-actin dependence of spindle orientation in synchronized cells. Wild-type (DLY1) cells were grown in YEPD at 30°C, synchronized by the addition of 25 ng/ml α-factor for 3 h, harvested, and resuspended in fresh YEPD. At 30, 45, and 60 min after release from α-factor, 6.25 μM Lat-A (or DMSO for controls) was added to aliquots of cells, which were then fixed at 75 min and processed to visualize tubulin. A, Kinetics of bud formation after release from α-factor. B, Quantification of preanaphase spindle orientation (scored as described in Materials and Methods). White bars, control; black bars, Lat-A treated. C, Quantification of postanaphase spindle orientation. White bars, control; black bars, Lat-A treated. D, Quantification of Lat-A induced errors observed in preanaphase (white bars) and postanaphase (black bars) spindles. To derive the proportion of cells displaying Lat-A–induced spindle misorientation, we subtracted the percent of misoriented spindles in control cells from that in Lat-A treated cells from the same sample. At least 100 cells were scored for each sample.
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Related In: Results  -  Collection

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Figure 4: F-actin dependence of spindle orientation in synchronized cells. Wild-type (DLY1) cells were grown in YEPD at 30°C, synchronized by the addition of 25 ng/ml α-factor for 3 h, harvested, and resuspended in fresh YEPD. At 30, 45, and 60 min after release from α-factor, 6.25 μM Lat-A (or DMSO for controls) was added to aliquots of cells, which were then fixed at 75 min and processed to visualize tubulin. A, Kinetics of bud formation after release from α-factor. B, Quantification of preanaphase spindle orientation (scored as described in Materials and Methods). White bars, control; black bars, Lat-A treated. C, Quantification of postanaphase spindle orientation. White bars, control; black bars, Lat-A treated. D, Quantification of Lat-A induced errors observed in preanaphase (white bars) and postanaphase (black bars) spindles. To derive the proportion of cells displaying Lat-A–induced spindle misorientation, we subtracted the percent of misoriented spindles in control cells from that in Lat-A treated cells from the same sample. At least 100 cells were scored for each sample.
Mentions: To examine whether cells traversing the cell cycle require actin to establish and/or maintain spindle orientation, a proliferating asynchronous population of wild-type cells was treated for 30 min with Lat-A. This induced a dramatic spindle misorientation in 53% of the cells (Fig. 3). Misaligned preanaphase spindles were observed in small- and medium-budded cells, but at lower frequency in large-budded cells (see below, Fig. 4). In addition, postanaphase spindles in medium-budded cells were often misaligned, probably resulting from elongation of short spindles that had become misoriented soon after Lat-A addition (Fig. 3 A; we return to this point in the Discussion). Thus, spindle orientation is actin-dependent in some fraction of a proliferating cell population, but not in hydroxyurea-arrested cells.

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