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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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Protein–protein interactions of Arp2/3 complex regulators. (A) The indicated regions of Wsp1p were tested for interaction with Myo1p and Vrp1p by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity measured in relative light units. (B) Approximately equal amounts of GST (lane 2, bottom) and GST–Myo1p(1077–1218) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Wsp1p-a (amino acids 1–497) (top) or Wsp1p-b (amino acids 1–283) (middle). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by fluorography (top two panels) or Coomassie staining (bottom). Only relevant portions of the Coomassie-stained gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lane 1 contains 10% of the input into the reactions. (C) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Vrp1p(1–309) (lane 3, bottom) bound to amylose beads were mixed with in vitro–translated Ws1p-b (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B. (D) A graphic representation of Cdc15p. Amino acid residues at the borders of known domains are shown. (E) The indicated regions of Myo1p were tested for interaction with Cdc15p (amino acids 1–282) by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity. (F) GST (lanes 2 and 4) or GST–Cdc15p(1–405) (lanes 3 and 5) bound to glutathione beads were incubated with protein lysates from a myo1-HA strain (KGY3960) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Lane 1 contained lysate from myo1-HA strain and is an input control. (G) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Myo1-a (amino acids 727–1041) (lanes 3 and 5, bottom) bound to amylose beads were mixed with either soluble GST–Cdc15p(1–405) (lanes 2 and 3) or GST–Sid4p (lane 5). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the GST fusion proteins before the binding reactions. (H) Approximately equal amounts of GST (lane 2, bottom) and GST–Cdc15p(1–405) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Myo1-a (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B.
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fig2: Protein–protein interactions of Arp2/3 complex regulators. (A) The indicated regions of Wsp1p were tested for interaction with Myo1p and Vrp1p by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity measured in relative light units. (B) Approximately equal amounts of GST (lane 2, bottom) and GST–Myo1p(1077–1218) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Wsp1p-a (amino acids 1–497) (top) or Wsp1p-b (amino acids 1–283) (middle). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by fluorography (top two panels) or Coomassie staining (bottom). Only relevant portions of the Coomassie-stained gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lane 1 contains 10% of the input into the reactions. (C) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Vrp1p(1–309) (lane 3, bottom) bound to amylose beads were mixed with in vitro–translated Ws1p-b (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B. (D) A graphic representation of Cdc15p. Amino acid residues at the borders of known domains are shown. (E) The indicated regions of Myo1p were tested for interaction with Cdc15p (amino acids 1–282) by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity. (F) GST (lanes 2 and 4) or GST–Cdc15p(1–405) (lanes 3 and 5) bound to glutathione beads were incubated with protein lysates from a myo1-HA strain (KGY3960) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Lane 1 contained lysate from myo1-HA strain and is an input control. (G) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Myo1-a (amino acids 727–1041) (lanes 3 and 5, bottom) bound to amylose beads were mixed with either soluble GST–Cdc15p(1–405) (lanes 2 and 3) or GST–Sid4p (lane 5). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the GST fusion proteins before the binding reactions. (H) Approximately equal amounts of GST (lane 2, bottom) and GST–Cdc15p(1–405) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Myo1-a (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B.

Mentions: In S. cerevisiae, Vrp1p, Las17p/Bee1p, and type I myosins form an Arp2/3 regulatory complex, with direct protein–protein interactions observed between Vrp1p and Las17p/Bee1p and between both of these proteins and the type I myosins (Evangelista et al., 2000; Lechler et al., 2000). Therefore, we asked whether the S. pombe homologues of these proteins also interact with one another. Similar to observations in S. cerevisiae, Wsp1p interacted strongly with the SH3 domain of Myo1p (residues 1077–1218) as well as with the COOH terminus of Vrp1p, by two hybrid analysis (Fig. 2 A). Further, GST–Myo1p(1077–1218) and maltose binding protein (MBP)–Vrp1p(206–309) fusion proteins bound directly to Wsp1p fragments produced in a coupled transcription/translation in vitro system (Fig. 2, B and C). These binding regions corresponded to those found to interact in the S. cerevisiae homologues (Evangelista et al., 2000; Lechler et al., 2000). Contrary to expectations, however, we found no evidence for an interaction between any regions of Myo1p and Vrp1p (unpublished data). Additionally, whereas vrp1Δ wsp1Δ cells were viable (unpublished data), deletion of both vrp1 and myo1 was synthetically lethal. Previous work has indicated that Wsp1p and Myo1p represent redundant pathways of Arp2/3 complex activation in S. pombe (Lee et al., 2000). Our data support this model and suggest that Vrp1p is most important for the Wsp1p pathway.


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)

Protein–protein interactions of Arp2/3 complex regulators. (A) The indicated regions of Wsp1p were tested for interaction with Myo1p and Vrp1p by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity measured in relative light units. (B) Approximately equal amounts of GST (lane 2, bottom) and GST–Myo1p(1077–1218) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Wsp1p-a (amino acids 1–497) (top) or Wsp1p-b (amino acids 1–283) (middle). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by fluorography (top two panels) or Coomassie staining (bottom). Only relevant portions of the Coomassie-stained gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lane 1 contains 10% of the input into the reactions. (C) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Vrp1p(1–309) (lane 3, bottom) bound to amylose beads were mixed with in vitro–translated Ws1p-b (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B. (D) A graphic representation of Cdc15p. Amino acid residues at the borders of known domains are shown. (E) The indicated regions of Myo1p were tested for interaction with Cdc15p (amino acids 1–282) by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity. (F) GST (lanes 2 and 4) or GST–Cdc15p(1–405) (lanes 3 and 5) bound to glutathione beads were incubated with protein lysates from a myo1-HA strain (KGY3960) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Lane 1 contained lysate from myo1-HA strain and is an input control. (G) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Myo1-a (amino acids 727–1041) (lanes 3 and 5, bottom) bound to amylose beads were mixed with either soluble GST–Cdc15p(1–405) (lanes 2 and 3) or GST–Sid4p (lane 5). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the GST fusion proteins before the binding reactions. (H) Approximately equal amounts of GST (lane 2, bottom) and GST–Cdc15p(1–405) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Myo1-a (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B.
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fig2: Protein–protein interactions of Arp2/3 complex regulators. (A) The indicated regions of Wsp1p were tested for interaction with Myo1p and Vrp1p by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity measured in relative light units. (B) Approximately equal amounts of GST (lane 2, bottom) and GST–Myo1p(1077–1218) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Wsp1p-a (amino acids 1–497) (top) or Wsp1p-b (amino acids 1–283) (middle). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by fluorography (top two panels) or Coomassie staining (bottom). Only relevant portions of the Coomassie-stained gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lane 1 contains 10% of the input into the reactions. (C) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Vrp1p(1–309) (lane 3, bottom) bound to amylose beads were mixed with in vitro–translated Ws1p-b (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B. (D) A graphic representation of Cdc15p. Amino acid residues at the borders of known domains are shown. (E) The indicated regions of Myo1p were tested for interaction with Cdc15p (amino acids 1–282) by two-hybrid analysis. LEU+ TRP+ transformants were tested for growth on selective media (not depicted) and assayed for β-galactosidase activity. (F) GST (lanes 2 and 4) or GST–Cdc15p(1–405) (lanes 3 and 5) bound to glutathione beads were incubated with protein lysates from a myo1-HA strain (KGY3960) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Lane 1 contained lysate from myo1-HA strain and is an input control. (G) Approximately equal amounts of MBP (lane 2, bottom) and MBP–Myo1-a (amino acids 727–1041) (lanes 3 and 5, bottom) bound to amylose beads were mixed with either soluble GST–Cdc15p(1–405) (lanes 2 and 3) or GST–Sid4p (lane 5). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate equal loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the GST fusion proteins before the binding reactions. (H) Approximately equal amounts of GST (lane 2, bottom) and GST–Cdc15p(1–405) (lane 3, bottom) bound to glutathione beads were mixed with in vitro–translated Myo1-a (top). Beads were collected, washed, and eluted as described in the Materials and methods. Proteins were further analyzed as in B.
Mentions: In S. cerevisiae, Vrp1p, Las17p/Bee1p, and type I myosins form an Arp2/3 regulatory complex, with direct protein–protein interactions observed between Vrp1p and Las17p/Bee1p and between both of these proteins and the type I myosins (Evangelista et al., 2000; Lechler et al., 2000). Therefore, we asked whether the S. pombe homologues of these proteins also interact with one another. Similar to observations in S. cerevisiae, Wsp1p interacted strongly with the SH3 domain of Myo1p (residues 1077–1218) as well as with the COOH terminus of Vrp1p, by two hybrid analysis (Fig. 2 A). Further, GST–Myo1p(1077–1218) and maltose binding protein (MBP)–Vrp1p(206–309) fusion proteins bound directly to Wsp1p fragments produced in a coupled transcription/translation in vitro system (Fig. 2, B and C). These binding regions corresponded to those found to interact in the S. cerevisiae homologues (Evangelista et al., 2000; Lechler et al., 2000). Contrary to expectations, however, we found no evidence for an interaction between any regions of Myo1p and Vrp1p (unpublished data). Additionally, whereas vrp1Δ wsp1Δ cells were viable (unpublished data), deletion of both vrp1 and myo1 was synthetically lethal. Previous work has indicated that Wsp1p and Myo1p represent redundant pathways of Arp2/3 complex activation in S. pombe (Lee et al., 2000). Our data support this model and suggest that Vrp1p is most important for the Wsp1p pathway.

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