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UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II.

Lord M, Pollard TD - J. Cell Biol. (2004)

Bottom Line: Thus, Rng3p contributes directly to the motility activity of native Myo2.Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring.In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

ABSTRACT
We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

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Effects of Rng3p repression or overexpression on Myo2p and actin localization and cytokinesis. Fluorescence and DIC micrographs of yeast cells expressing GFP-Myo2p and stained for actin with rhodamine phalloidin. Bars, 5 μm. Cells were mounted on 25% gelatin pads in the appropriate EMM medium. In a separate experiment, cells were treated with Hoescht stain to mark nuclei. (A) Repression of Rng3p expression: MLP 640 (41nmt1 promoter-rng3, GFP-myo2) was grown in liquid EMM medium with 5 μg/ml thiamine to repress Rng3p expression (left micrographs) or without thiamine (right micrographs). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either nonrepressing (− thiamine) or repressing (+ thiamine) conditions as quantitated by scoring nuclei/cell using fluorescence microscopy. (B) Overexpression of Rng3p and (C) overexpression of the Rng3p UCS domain. MLP 639 (3nmt1 promoter-rng3, GFP-myo2) and FY 435 carrying pGST-rng3-UCS were grown in liquid EMM medium supplemented with 5 μg/ml thiamine (to repress Rng3p and Rng3p-UCS expression to a modest level) or without thiamine (to overexpress Rng3p and Rng3p-UCS). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either inducing (− thiamine) or uninducing (+ thiamine) conditions.
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fig4: Effects of Rng3p repression or overexpression on Myo2p and actin localization and cytokinesis. Fluorescence and DIC micrographs of yeast cells expressing GFP-Myo2p and stained for actin with rhodamine phalloidin. Bars, 5 μm. Cells were mounted on 25% gelatin pads in the appropriate EMM medium. In a separate experiment, cells were treated with Hoescht stain to mark nuclei. (A) Repression of Rng3p expression: MLP 640 (41nmt1 promoter-rng3, GFP-myo2) was grown in liquid EMM medium with 5 μg/ml thiamine to repress Rng3p expression (left micrographs) or without thiamine (right micrographs). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either nonrepressing (− thiamine) or repressing (+ thiamine) conditions as quantitated by scoring nuclei/cell using fluorescence microscopy. (B) Overexpression of Rng3p and (C) overexpression of the Rng3p UCS domain. MLP 639 (3nmt1 promoter-rng3, GFP-myo2) and FY 435 carrying pGST-rng3-UCS were grown in liquid EMM medium supplemented with 5 μg/ml thiamine (to repress Rng3p and Rng3p-UCS expression to a modest level) or without thiamine (to overexpress Rng3p and Rng3p-UCS). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either inducing (− thiamine) or uninducing (+ thiamine) conditions.

Mentions: Repression of Rng3p expression compromises concentration of Myo2 in the contractile ring and cytokinesis (Fig. 4 A), similar to a rng3 mutant (Wong et al., 2000). This experiment used a strain with two chromosomal replacements: a thiamine repressible 41nmt1 promoter replaced the rng3 promoter; and a GFP-myo2 fusion replaced the Myo2p coding sequence. Control cells expressing Rng3p in the absence of thiamine had no defects in cytokinesis or Myo2p/actin localization. Repression of rng3 expression with thiamine resulted in cells with Myo2 and actin mislocalized together in disorganized structures that failed to condense into contractile rings. Cytokinesis and septation failed, so these cells became multinucleate.


UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II.

Lord M, Pollard TD - J. Cell Biol. (2004)

Effects of Rng3p repression or overexpression on Myo2p and actin localization and cytokinesis. Fluorescence and DIC micrographs of yeast cells expressing GFP-Myo2p and stained for actin with rhodamine phalloidin. Bars, 5 μm. Cells were mounted on 25% gelatin pads in the appropriate EMM medium. In a separate experiment, cells were treated with Hoescht stain to mark nuclei. (A) Repression of Rng3p expression: MLP 640 (41nmt1 promoter-rng3, GFP-myo2) was grown in liquid EMM medium with 5 μg/ml thiamine to repress Rng3p expression (left micrographs) or without thiamine (right micrographs). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either nonrepressing (− thiamine) or repressing (+ thiamine) conditions as quantitated by scoring nuclei/cell using fluorescence microscopy. (B) Overexpression of Rng3p and (C) overexpression of the Rng3p UCS domain. MLP 639 (3nmt1 promoter-rng3, GFP-myo2) and FY 435 carrying pGST-rng3-UCS were grown in liquid EMM medium supplemented with 5 μg/ml thiamine (to repress Rng3p and Rng3p-UCS expression to a modest level) or without thiamine (to overexpress Rng3p and Rng3p-UCS). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either inducing (− thiamine) or uninducing (+ thiamine) conditions.
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fig4: Effects of Rng3p repression or overexpression on Myo2p and actin localization and cytokinesis. Fluorescence and DIC micrographs of yeast cells expressing GFP-Myo2p and stained for actin with rhodamine phalloidin. Bars, 5 μm. Cells were mounted on 25% gelatin pads in the appropriate EMM medium. In a separate experiment, cells were treated with Hoescht stain to mark nuclei. (A) Repression of Rng3p expression: MLP 640 (41nmt1 promoter-rng3, GFP-myo2) was grown in liquid EMM medium with 5 μg/ml thiamine to repress Rng3p expression (left micrographs) or without thiamine (right micrographs). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either nonrepressing (− thiamine) or repressing (+ thiamine) conditions as quantitated by scoring nuclei/cell using fluorescence microscopy. (B) Overexpression of Rng3p and (C) overexpression of the Rng3p UCS domain. MLP 639 (3nmt1 promoter-rng3, GFP-myo2) and FY 435 carrying pGST-rng3-UCS were grown in liquid EMM medium supplemented with 5 μg/ml thiamine (to repress Rng3p and Rng3p-UCS expression to a modest level) or without thiamine (to overexpress Rng3p and Rng3p-UCS). (top) GFP-Myo2p; (middle) actin; (bottom) DIC. Plots on the left summarize the phenotypes of cells grown in either inducing (− thiamine) or uninducing (+ thiamine) conditions.
Mentions: Repression of Rng3p expression compromises concentration of Myo2 in the contractile ring and cytokinesis (Fig. 4 A), similar to a rng3 mutant (Wong et al., 2000). This experiment used a strain with two chromosomal replacements: a thiamine repressible 41nmt1 promoter replaced the rng3 promoter; and a GFP-myo2 fusion replaced the Myo2p coding sequence. Control cells expressing Rng3p in the absence of thiamine had no defects in cytokinesis or Myo2p/actin localization. Repression of rng3 expression with thiamine resulted in cells with Myo2 and actin mislocalized together in disorganized structures that failed to condense into contractile rings. Cytokinesis and septation failed, so these cells became multinucleate.

Bottom Line: Thus, Rng3p contributes directly to the motility activity of native Myo2.Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring.In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

ABSTRACT
We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

Show MeSH
Related in: MedlinePlus