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A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast.

Lechler T, Jonsdottir GA, Klee SK, Pellman D, Li R - J. Cell Biol. (2001)

Bottom Line: One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides.The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation.Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

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Polarized Bee1-GFP defines the site of actin polymerization in vivo. (A) A population enriched in polarized unbudded cells was generated by releasing G1-arrested cells into the cell cycle in the presence of Lat-A. 1 h after release, Lat-A was washed away, and cells were fixed at various time points. The cells were stained for Bee1-GFP (anti-GFP antibody) and F-actin (rhodamine phalloidin). Shown are cells from the 5 min time point. (B) Quantitation of results from the experiment described in A. At various time points after Lat-A washout, the percentage of cells with F-actin colocalizing with polarized Bee1-GFP was determined. Only cells with polarized Bee1-GFP were counted.
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fig3: Polarized Bee1-GFP defines the site of actin polymerization in vivo. (A) A population enriched in polarized unbudded cells was generated by releasing G1-arrested cells into the cell cycle in the presence of Lat-A. 1 h after release, Lat-A was washed away, and cells were fixed at various time points. The cells were stained for Bee1-GFP (anti-GFP antibody) and F-actin (rhodamine phalloidin). Shown are cells from the 5 min time point. (B) Quantitation of results from the experiment described in A. At various time points after Lat-A washout, the percentage of cells with F-actin colocalizing with polarized Bee1-GFP was determined. Only cells with polarized Bee1-GFP were counted.

Mentions: Results described above suggest that Cdc42p induces actin cytoskeleton polarization in part through polarized recruitment of the Bee1–Vrp1 complex, which then locally activates the Arp2/3 complex. Under this hypothesis, actin polymerization should occur at the site of Bee1p/Vrp1p recruitment upon washout of Lat-A in the experiments described in Fig. 2. Therefore, we examined the regeneration of F-actin in cells that have recruited Bee1p–Vrp1p complex to the presumptive bud site. cdc28-13 cells expressing Bee1-GFP were released from a G1 arrest in the presence of Lat-A. After 1 h, Lat-A was washed out, and cells were fixed at varying times and stained for both F-actin and Bee1-GFP. At the zero time point after washout, no actin structures were visible in cells, but many contained polarized Bee1-GFP. By 1 min after washout, a small fraction of cells contained very low but detectable levels of F-actin. In all cases, this actin colocalized with Bee1-GFP (Fig. 3). By the 2 min time point, most cells had polarized Bee1-GFP that colocalized with F-actin, although the levels of F-actin were still low compared with cells without Lat-A treatment. This result suggests that cortical actin assembly is initiated at the site of Bee1p/Vrp1p recruitment. As a control, when the same experiment was done in a cdc24-1 mutant background in which there is no Bee1p polarization we did not observe the regeneration of F-actin at the presumptive bud site, but rather we observed the regeneration in a depolarized manner (unpublished data).


A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast.

Lechler T, Jonsdottir GA, Klee SK, Pellman D, Li R - J. Cell Biol. (2001)

Polarized Bee1-GFP defines the site of actin polymerization in vivo. (A) A population enriched in polarized unbudded cells was generated by releasing G1-arrested cells into the cell cycle in the presence of Lat-A. 1 h after release, Lat-A was washed away, and cells were fixed at various time points. The cells were stained for Bee1-GFP (anti-GFP antibody) and F-actin (rhodamine phalloidin). Shown are cells from the 5 min time point. (B) Quantitation of results from the experiment described in A. At various time points after Lat-A washout, the percentage of cells with F-actin colocalizing with polarized Bee1-GFP was determined. Only cells with polarized Bee1-GFP were counted.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198833&req=5

fig3: Polarized Bee1-GFP defines the site of actin polymerization in vivo. (A) A population enriched in polarized unbudded cells was generated by releasing G1-arrested cells into the cell cycle in the presence of Lat-A. 1 h after release, Lat-A was washed away, and cells were fixed at various time points. The cells were stained for Bee1-GFP (anti-GFP antibody) and F-actin (rhodamine phalloidin). Shown are cells from the 5 min time point. (B) Quantitation of results from the experiment described in A. At various time points after Lat-A washout, the percentage of cells with F-actin colocalizing with polarized Bee1-GFP was determined. Only cells with polarized Bee1-GFP were counted.
Mentions: Results described above suggest that Cdc42p induces actin cytoskeleton polarization in part through polarized recruitment of the Bee1–Vrp1 complex, which then locally activates the Arp2/3 complex. Under this hypothesis, actin polymerization should occur at the site of Bee1p/Vrp1p recruitment upon washout of Lat-A in the experiments described in Fig. 2. Therefore, we examined the regeneration of F-actin in cells that have recruited Bee1p–Vrp1p complex to the presumptive bud site. cdc28-13 cells expressing Bee1-GFP were released from a G1 arrest in the presence of Lat-A. After 1 h, Lat-A was washed out, and cells were fixed at varying times and stained for both F-actin and Bee1-GFP. At the zero time point after washout, no actin structures were visible in cells, but many contained polarized Bee1-GFP. By 1 min after washout, a small fraction of cells contained very low but detectable levels of F-actin. In all cases, this actin colocalized with Bee1-GFP (Fig. 3). By the 2 min time point, most cells had polarized Bee1-GFP that colocalized with F-actin, although the levels of F-actin were still low compared with cells without Lat-A treatment. This result suggests that cortical actin assembly is initiated at the site of Bee1p/Vrp1p recruitment. As a control, when the same experiment was done in a cdc24-1 mutant background in which there is no Bee1p polarization we did not observe the regeneration of F-actin at the presumptive bud site, but rather we observed the regeneration in a depolarized manner (unpublished data).

Bottom Line: One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides.The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation.Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

Show MeSH
Related in: MedlinePlus