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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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Cdc15p is required for formation of the medial actin ring. (A) Spores from the heterozygous cdc15+/cdc15::ura4+ diploid were released into selective media (−uracil), indicated by +, or nonselective media (YE), indicated by −, and after 16 h were fixed and stained with AlexaFluor 488–phalloidin. The presence of a CAR in germinated cells was determined in binucleates and mononucleates. (B) A representative image of a binucleate cdc15::ura4 cell, fixed and stained with AlexaFluor 488–phalloidin to visualize actin (green) and DAPI to visualize DNA (blue). (C) Myo1p–GFP localization in G2-arrested cdc25-22 cells either expressing (induced) or not expressing (uninduced) nmt1cdc15.
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fig6: Cdc15p is required for formation of the medial actin ring. (A) Spores from the heterozygous cdc15+/cdc15::ura4+ diploid were released into selective media (−uracil), indicated by +, or nonselective media (YE), indicated by −, and after 16 h were fixed and stained with AlexaFluor 488–phalloidin. The presence of a CAR in germinated cells was determined in binucleates and mononucleates. (B) A representative image of a binucleate cdc15::ura4 cell, fixed and stained with AlexaFluor 488–phalloidin to visualize actin (green) and DAPI to visualize DNA (blue). (C) Myo1p–GFP localization in G2-arrested cdc25-22 cells either expressing (induced) or not expressing (uninduced) nmt1cdc15.

Mentions: Cdc15p is required for medial actin ring formation. Recent work has established that formins represent an Arp2/3 complex–independent pathway for the nucleation of actin filaments (Evangelista et al., 2002; Pruyne et al., 2002; Sagot et al., 2002). Furthermore, formation of the CAR in S. pombe depends upon both the Arp2/3 complex and Cdc12p (Pelham and Chang, 2002). As Cdc15p is involved in recruitment of both actin nucleation pathways required for CAR formation, we predicted that in the absence of Cdc15p function, actin rings should not be able to form. Contrary to this prediction, however, actin rings were reported to form in cdc15-140 cells (Balasubramanian et al., 1998). Re-examination of this issue indicated that although rings could be detected after 4 h incubation at the restrictive temperature of 36°C in cdc15-140 cells, this occurred in <2% of cells (vs. 12% in a similarly treated wild-type culture), and in accordance with another report on CAR formation in S. pombe (Arai and Mabuchi, 2002), these rings were poorly organized and incomplete (unpublished data). We also examined CAR formation in cdc15::ura4 cells. Spores from a cdc15+/cdc15::ura4+ heterozygous diploid were inoculated into liquid medium either with or without selection for Ura4+ growth. Under selective conditions, no CARs were detected as spores germinated and underwent their first mitosis (Fig. 6, A and B). Rather, actin patches remained primarily at cell tips. This is in contrast to spores released into nonselective medium, where CARs were observed in ∼50% of binucleate cells and a significant portion of germinated mononucleate cells (Fig. 6 A), as would be predicted.


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)

Cdc15p is required for formation of the medial actin ring. (A) Spores from the heterozygous cdc15+/cdc15::ura4+ diploid were released into selective media (−uracil), indicated by +, or nonselective media (YE), indicated by −, and after 16 h were fixed and stained with AlexaFluor 488–phalloidin. The presence of a CAR in germinated cells was determined in binucleates and mononucleates. (B) A representative image of a binucleate cdc15::ura4 cell, fixed and stained with AlexaFluor 488–phalloidin to visualize actin (green) and DAPI to visualize DNA (blue). (C) Myo1p–GFP localization in G2-arrested cdc25-22 cells either expressing (induced) or not expressing (uninduced) nmt1cdc15.
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Related In: Results  -  Collection

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

fig6: Cdc15p is required for formation of the medial actin ring. (A) Spores from the heterozygous cdc15+/cdc15::ura4+ diploid were released into selective media (−uracil), indicated by +, or nonselective media (YE), indicated by −, and after 16 h were fixed and stained with AlexaFluor 488–phalloidin. The presence of a CAR in germinated cells was determined in binucleates and mononucleates. (B) A representative image of a binucleate cdc15::ura4 cell, fixed and stained with AlexaFluor 488–phalloidin to visualize actin (green) and DAPI to visualize DNA (blue). (C) Myo1p–GFP localization in G2-arrested cdc25-22 cells either expressing (induced) or not expressing (uninduced) nmt1cdc15.
Mentions: Cdc15p is required for medial actin ring formation. Recent work has established that formins represent an Arp2/3 complex–independent pathway for the nucleation of actin filaments (Evangelista et al., 2002; Pruyne et al., 2002; Sagot et al., 2002). Furthermore, formation of the CAR in S. pombe depends upon both the Arp2/3 complex and Cdc12p (Pelham and Chang, 2002). As Cdc15p is involved in recruitment of both actin nucleation pathways required for CAR formation, we predicted that in the absence of Cdc15p function, actin rings should not be able to form. Contrary to this prediction, however, actin rings were reported to form in cdc15-140 cells (Balasubramanian et al., 1998). Re-examination of this issue indicated that although rings could be detected after 4 h incubation at the restrictive temperature of 36°C in cdc15-140 cells, this occurred in <2% of cells (vs. 12% in a similarly treated wild-type culture), and in accordance with another report on CAR formation in S. pombe (Arai and Mabuchi, 2002), these rings were poorly organized and incomplete (unpublished data). We also examined CAR formation in cdc15::ura4 cells. Spores from a cdc15+/cdc15::ura4+ heterozygous diploid were inoculated into liquid medium either with or without selection for Ura4+ growth. Under selective conditions, no CARs were detected as spores germinated and underwent their first mitosis (Fig. 6, A and B). Rather, actin patches remained primarily at cell tips. This is in contrast to spores released into nonselective medium, where CARs were observed in ∼50% of binucleate cells and a significant portion of germinated mononucleate cells (Fig. 6 A), as would be predicted.

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