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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 interacts directly with Cdc12p. (A) The indicated regions of Cdc12p 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 measured in relative light units. GFP fusions of the cdc12 constructs were also expressed under control of the nmt81 promoter and visually examined for their ability (+) or lack of ability (−) to localize to an interphase spot structure and also to the CAR (column 3). (B) MBP (lane 2) or MBP–Cdc12p-b (amino acids 1–764) (lanes 3 and 5) both bound to amylose beads were incubated with protein lysates from either a cdc15-HA strain (KGY3020) or a cut9-HA strain (KGY1463) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Only relevant portions of the gels are shown; however, all proteins ran at the predicted sizes. (C) GST (lane 2, bottom) or GST–Cdc12-b (lanes 3 and 5, bottom) both bound to glutathione beads were mixed with either soluble MBP–Cdc15p(1–282) (lanes 1–3, top) or soluble MBP–Cdc11p(1–660) (lanes 4 and 5, top). Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the MBP fusion proteins before the binding reactions. (D) Localization of Cdc15p–GFP in cdc15-GFP cdc12-112 cells after 4 h at 36°C. (E) Phase contrast image of cdc15-140 cdc12-299 cells grown at 25°C. Bars, 5 μm.
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fig5: Cdc15p interacts directly with Cdc12p. (A) The indicated regions of Cdc12p 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 measured in relative light units. GFP fusions of the cdc12 constructs were also expressed under control of the nmt81 promoter and visually examined for their ability (+) or lack of ability (−) to localize to an interphase spot structure and also to the CAR (column 3). (B) MBP (lane 2) or MBP–Cdc12p-b (amino acids 1–764) (lanes 3 and 5) both bound to amylose beads were incubated with protein lysates from either a cdc15-HA strain (KGY3020) or a cut9-HA strain (KGY1463) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Only relevant portions of the gels are shown; however, all proteins ran at the predicted sizes. (C) GST (lane 2, bottom) or GST–Cdc12-b (lanes 3 and 5, bottom) both bound to glutathione beads were mixed with either soluble MBP–Cdc15p(1–282) (lanes 1–3, top) or soluble MBP–Cdc11p(1–660) (lanes 4 and 5, top). Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the MBP fusion proteins before the binding reactions. (D) Localization of Cdc15p–GFP in cdc15-GFP cdc12-112 cells after 4 h at 36°C. (E) Phase contrast image of cdc15-140 cdc12-299 cells grown at 25°C. Bars, 5 μm.

Mentions: Given the colocalization of Cdc15p and Cdc12p to both the CAR and the interphase spot, we asked if these proteins could interact. By two-hybrid analysis, a strong interaction was detected between the NH2 terminus of Cdc15p(1–282) and NH2-terminal constructs of Cdc12p that included its formin homology 3 (FH3) domain (Fig. 5 A). Previous work has suggested that FH3 domains of formins are involved in targeting them to discrete locations within S. pombe cells (Petersen et al., 1998). The NH2 terminus and FH3 domain of Cdc12p suffice not only for its localizations to the CAR and the motile spot, but also for its interaction with Cdc15p (Fig. 5 A). Notably, the COOH-terminal SH3 domain of Cdc15p is not involved in its interaction with Cdc12p. Indeed, a construct of Cdc15p lacking its SH3 domain is able to rescue cdc15 cells, indicating that it does not play an essential role in Cdc15p function during cytokinesis (unpublished data).


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 interacts directly with Cdc12p. (A) The indicated regions of Cdc12p 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 measured in relative light units. GFP fusions of the cdc12 constructs were also expressed under control of the nmt81 promoter and visually examined for their ability (+) or lack of ability (−) to localize to an interphase spot structure and also to the CAR (column 3). (B) MBP (lane 2) or MBP–Cdc12p-b (amino acids 1–764) (lanes 3 and 5) both bound to amylose beads were incubated with protein lysates from either a cdc15-HA strain (KGY3020) or a cut9-HA strain (KGY1463) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Only relevant portions of the gels are shown; however, all proteins ran at the predicted sizes. (C) GST (lane 2, bottom) or GST–Cdc12-b (lanes 3 and 5, bottom) both bound to glutathione beads were mixed with either soluble MBP–Cdc15p(1–282) (lanes 1–3, top) or soluble MBP–Cdc11p(1–660) (lanes 4 and 5, top). Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the MBP fusion proteins before the binding reactions. (D) Localization of Cdc15p–GFP in cdc15-GFP cdc12-112 cells after 4 h at 36°C. (E) Phase contrast image of cdc15-140 cdc12-299 cells grown at 25°C. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172828&req=5

fig5: Cdc15p interacts directly with Cdc12p. (A) The indicated regions of Cdc12p 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 measured in relative light units. GFP fusions of the cdc12 constructs were also expressed under control of the nmt81 promoter and visually examined for their ability (+) or lack of ability (−) to localize to an interphase spot structure and also to the CAR (column 3). (B) MBP (lane 2) or MBP–Cdc12p-b (amino acids 1–764) (lanes 3 and 5) both bound to amylose beads were incubated with protein lysates from either a cdc15-HA strain (KGY3020) or a cut9-HA strain (KGY1463) and subsequently extensively washed in binding buffer. Bound proteins were then divided and analyzed by immunoblotting (top) and Coomassie staining (bottom). Only relevant portions of the gels are shown; however, all proteins ran at the predicted sizes. (C) GST (lane 2, bottom) or GST–Cdc12-b (lanes 3 and 5, bottom) both bound to glutathione beads were mixed with either soluble MBP–Cdc15p(1–282) (lanes 1–3, top) or soluble MBP–Cdc11p(1–660) (lanes 4 and 5, top). Proteins were resolved by SDS-PAGE and detected by Coomassie staining. Only relevant portions of the Coomassie gel are shown to indicate loading; however, all proteins ran at the predicted sizes. Lanes 1 and 4 contain samples of the MBP fusion proteins before the binding reactions. (D) Localization of Cdc15p–GFP in cdc15-GFP cdc12-112 cells after 4 h at 36°C. (E) Phase contrast image of cdc15-140 cdc12-299 cells grown at 25°C. Bars, 5 μm.
Mentions: Given the colocalization of Cdc15p and Cdc12p to both the CAR and the interphase spot, we asked if these proteins could interact. By two-hybrid analysis, a strong interaction was detected between the NH2 terminus of Cdc15p(1–282) and NH2-terminal constructs of Cdc12p that included its formin homology 3 (FH3) domain (Fig. 5 A). Previous work has suggested that FH3 domains of formins are involved in targeting them to discrete locations within S. pombe cells (Petersen et al., 1998). The NH2 terminus and FH3 domain of Cdc12p suffice not only for its localizations to the CAR and the motile spot, but also for its interaction with Cdc15p (Fig. 5 A). Notably, the COOH-terminal SH3 domain of Cdc15p is not involved in its interaction with Cdc12p. Indeed, a construct of Cdc15p lacking its SH3 domain is able to rescue cdc15 cells, indicating that it does not play an essential role in Cdc15p function during cytokinesis (unpublished data).

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