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The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe.

Carnahan RH, Gould KL - J. Cell Biol. (2003)

Bottom Line: Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants.Cdc15p also binds directly to Cdc12p.We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

ABSTRACT
Cytokinetic actin ring (CAR) formation in Schizosaccharomyces pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the S. pombe Cdc15 homology family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring-nucleating structure before CAR formation. The disruption of these interactions in cdc15 cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

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GFP-tagged Arp2/3 activators fail to localize to the medial region in a cdc15 mutant at the restrictive temperature. (A–C) cdc15-140 cells with either wsp1-GFP (KGY358), vrp1-GFP (KGY108), or myo1-GFP (KGY3963) alleles were grown to log phase at 25°C and shifted to 36°C for 4 h before live cells were imaged. Bar, 5 μm. (D) myo1-GFP cdc15-140 cells (KGY3963) were synchronized by lactose gradients and released to 25°C or 36°C. Live cells were monitored for Myo1p–GFP, and parallel samples were fixed and stained with DAPI. Localization of Myo1p to the medial region is indicated by triangles, and cells with two or more nuclei are indicated by squares.
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fig1: GFP-tagged Arp2/3 activators fail to localize to the medial region in a cdc15 mutant at the restrictive temperature. (A–C) cdc15-140 cells with either wsp1-GFP (KGY358), vrp1-GFP (KGY108), or myo1-GFP (KGY3963) alleles were grown to log phase at 25°C and shifted to 36°C for 4 h before live cells were imaged. Bar, 5 μm. (D) myo1-GFP cdc15-140 cells (KGY3963) were synchronized by lactose gradients and released to 25°C or 36°C. Live cells were monitored for Myo1p–GFP, and parallel samples were fixed and stained with DAPI. Localization of Myo1p to the medial region is indicated by triangles, and cells with two or more nuclei are indicated by squares.

Mentions: We showed previously that neither actin patches nor the Arp2/3 complex are recruited to the medial region of cdc15-140 cells grown at its restrictive temperature (Balasubramanian et al., 1998). We therefore asked whether activators of the Arp2/3 complex are also not properly localized in this mutant. We examined in live cells the localization of endogenously GFP-tagged Myo1p and Wsp1p, as well as, verprolin (Vrp1p), a potential regulator of these activators. Consistent with known roles in actin patch regulation (Lee et al., 2000; Naqvi et al., 2001; Toya et al., 2001), Myo1p–GFP, Wsp1p–GFP, and Vrp1p–GFP were all localized as patches at growing cell ends during interphase, whereas in mitosis, they were detected in the medial region of cells (Fig. 1, A–C). In cdc15-140 cells at 36°C, however, all three proteins were observed at the cell cortex and failed to localize to the medial region of cells (Fig. 1, A–C). Examination of cdc15-140 myo1-GFP cells, synchronized in G2 and released to either permissive or restrictive temperature, revealed that Myo1p was never detected in the medial region in the absence of Cdc15p function (Fig. 1 D). Similar results were obtained for Wsp1p–GFP and Vrp1p–GFP (not depicted). We conclude that Cdc15p function is required for medially directed mitotic recruitment of not only the Arp2/3 complex, but also of its known regulators.


The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe.

Carnahan RH, Gould KL - J. Cell Biol. (2003)

GFP-tagged Arp2/3 activators fail to localize to the medial region in a cdc15 mutant at the restrictive temperature. (A–C) cdc15-140 cells with either wsp1-GFP (KGY358), vrp1-GFP (KGY108), or myo1-GFP (KGY3963) alleles were grown to log phase at 25°C and shifted to 36°C for 4 h before live cells were imaged. Bar, 5 μm. (D) myo1-GFP cdc15-140 cells (KGY3963) were synchronized by lactose gradients and released to 25°C or 36°C. Live cells were monitored for Myo1p–GFP, and parallel samples were fixed and stained with DAPI. Localization of Myo1p to the medial region is indicated by triangles, and cells with two or more nuclei are indicated by squares.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172828&req=5

fig1: GFP-tagged Arp2/3 activators fail to localize to the medial region in a cdc15 mutant at the restrictive temperature. (A–C) cdc15-140 cells with either wsp1-GFP (KGY358), vrp1-GFP (KGY108), or myo1-GFP (KGY3963) alleles were grown to log phase at 25°C and shifted to 36°C for 4 h before live cells were imaged. Bar, 5 μm. (D) myo1-GFP cdc15-140 cells (KGY3963) were synchronized by lactose gradients and released to 25°C or 36°C. Live cells were monitored for Myo1p–GFP, and parallel samples were fixed and stained with DAPI. Localization of Myo1p to the medial region is indicated by triangles, and cells with two or more nuclei are indicated by squares.
Mentions: We showed previously that neither actin patches nor the Arp2/3 complex are recruited to the medial region of cdc15-140 cells grown at its restrictive temperature (Balasubramanian et al., 1998). We therefore asked whether activators of the Arp2/3 complex are also not properly localized in this mutant. We examined in live cells the localization of endogenously GFP-tagged Myo1p and Wsp1p, as well as, verprolin (Vrp1p), a potential regulator of these activators. Consistent with known roles in actin patch regulation (Lee et al., 2000; Naqvi et al., 2001; Toya et al., 2001), Myo1p–GFP, Wsp1p–GFP, and Vrp1p–GFP were all localized as patches at growing cell ends during interphase, whereas in mitosis, they were detected in the medial region of cells (Fig. 1, A–C). In cdc15-140 cells at 36°C, however, all three proteins were observed at the cell cortex and failed to localize to the medial region of cells (Fig. 1, A–C). Examination of cdc15-140 myo1-GFP cells, synchronized in G2 and released to either permissive or restrictive temperature, revealed that Myo1p was never detected in the medial region in the absence of Cdc15p function (Fig. 1 D). Similar results were obtained for Wsp1p–GFP and Vrp1p–GFP (not depicted). We conclude that Cdc15p function is required for medially directed mitotic recruitment of not only the Arp2/3 complex, but also of its known regulators.

Bottom Line: Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants.Cdc15p also binds directly to Cdc12p.We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

ABSTRACT
Cytokinetic actin ring (CAR) formation in Schizosaccharomyces pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the S. pombe Cdc15 homology family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring-nucleating structure before CAR formation. The disruption of these interactions in cdc15 cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

Show MeSH
Related in: MedlinePlus