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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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Rng3p colocalizes with Myo2p at the contractile ring. (A) Epi-fluorescence and DIC micrographs of live yeast cells expressing Rng3p-GFP3 (MLP 662) or Rng3p-YFP3 (MLP 660). (B–E) Spinning disk confocal fluorescence micrographs of live yeast cells expressing CFP-Myo2p and Rng3p-YFP3 (MLP 665). Bars, 5 μm. Cells were mounted on 25% gelatin pads in EMM medium and photographed with GFP, YFP, or CFP filters. (A) Images of Rng3p-GFP3 (left panels) and Rng3p-YFP3 (right panels) in dividing cells. (B) Images of Myo2p (left), Rng3p (middle), and their merge (right) in two cells from a stack of 12 confocal z sections of 0.6 μm. (C) Images of Myo2p, Rng3p, and their merge are shown (as in B). Asterisk denotes Myo2p broad band; arrowhead indicates Myo2p ring lacking Rng3p. (D and E) Time-lapse series of a cell at intervals of 15 min (D) and 2 min (E). To avoid bleaching a single z section was collected at each time point. Myo2p and Rng3p are shown in merged format. Myo2p is detected at the contractile ring (D, 15' panel) before Rng3p. Both constrict together (D and E; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). n, position of nuclei.
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fig5: Rng3p colocalizes with Myo2p at the contractile ring. (A) Epi-fluorescence and DIC micrographs of live yeast cells expressing Rng3p-GFP3 (MLP 662) or Rng3p-YFP3 (MLP 660). (B–E) Spinning disk confocal fluorescence micrographs of live yeast cells expressing CFP-Myo2p and Rng3p-YFP3 (MLP 665). Bars, 5 μm. Cells were mounted on 25% gelatin pads in EMM medium and photographed with GFP, YFP, or CFP filters. (A) Images of Rng3p-GFP3 (left panels) and Rng3p-YFP3 (right panels) in dividing cells. (B) Images of Myo2p (left), Rng3p (middle), and their merge (right) in two cells from a stack of 12 confocal z sections of 0.6 μm. (C) Images of Myo2p, Rng3p, and their merge are shown (as in B). Asterisk denotes Myo2p broad band; arrowhead indicates Myo2p ring lacking Rng3p. (D and E) Time-lapse series of a cell at intervals of 15 min (D) and 2 min (E). To avoid bleaching a single z section was collected at each time point. Myo2p and Rng3p are shown in merged format. Myo2p is detected at the contractile ring (D, 15' panel) before Rng3p. Both constrict together (D and E; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). n, position of nuclei.

Mentions: Rng3p-GFP3 and Rng3p-YFP3 concentrated in contractile rings from anaphase B through constriction (Fig. 5). CFP-Myo2p joins the contractile ring earlier, beginning with a broad band of small dots before condensing into the contractile ring (Wu et al., 2003). Thus, 21% of full-diameter CFP-Myo2p contractile rings lacked detectable Rng3p-YFP3, whereas both CFP-Myo2p and Rng3p-YFP3 concentrated in all constricting contractile rings (Fig. 5, C–E; note the positions of the nuclei in Fig. 5 D; Video 4, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). The Rng3p-YFP3 signal was weak, so we cannot rule out low concentrations of Rng3-YFP3 in immature contractile rings, but Rng3p lags behind Myo2p incorporation into the ring.


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

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

Rng3p colocalizes with Myo2p at the contractile ring. (A) Epi-fluorescence and DIC micrographs of live yeast cells expressing Rng3p-GFP3 (MLP 662) or Rng3p-YFP3 (MLP 660). (B–E) Spinning disk confocal fluorescence micrographs of live yeast cells expressing CFP-Myo2p and Rng3p-YFP3 (MLP 665). Bars, 5 μm. Cells were mounted on 25% gelatin pads in EMM medium and photographed with GFP, YFP, or CFP filters. (A) Images of Rng3p-GFP3 (left panels) and Rng3p-YFP3 (right panels) in dividing cells. (B) Images of Myo2p (left), Rng3p (middle), and their merge (right) in two cells from a stack of 12 confocal z sections of 0.6 μm. (C) Images of Myo2p, Rng3p, and their merge are shown (as in B). Asterisk denotes Myo2p broad band; arrowhead indicates Myo2p ring lacking Rng3p. (D and E) Time-lapse series of a cell at intervals of 15 min (D) and 2 min (E). To avoid bleaching a single z section was collected at each time point. Myo2p and Rng3p are shown in merged format. Myo2p is detected at the contractile ring (D, 15' panel) before Rng3p. Both constrict together (D and E; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). n, position of nuclei.
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fig5: Rng3p colocalizes with Myo2p at the contractile ring. (A) Epi-fluorescence and DIC micrographs of live yeast cells expressing Rng3p-GFP3 (MLP 662) or Rng3p-YFP3 (MLP 660). (B–E) Spinning disk confocal fluorescence micrographs of live yeast cells expressing CFP-Myo2p and Rng3p-YFP3 (MLP 665). Bars, 5 μm. Cells were mounted on 25% gelatin pads in EMM medium and photographed with GFP, YFP, or CFP filters. (A) Images of Rng3p-GFP3 (left panels) and Rng3p-YFP3 (right panels) in dividing cells. (B) Images of Myo2p (left), Rng3p (middle), and their merge (right) in two cells from a stack of 12 confocal z sections of 0.6 μm. (C) Images of Myo2p, Rng3p, and their merge are shown (as in B). Asterisk denotes Myo2p broad band; arrowhead indicates Myo2p ring lacking Rng3p. (D and E) Time-lapse series of a cell at intervals of 15 min (D) and 2 min (E). To avoid bleaching a single z section was collected at each time point. Myo2p and Rng3p are shown in merged format. Myo2p is detected at the contractile ring (D, 15' panel) before Rng3p. Both constrict together (D and E; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). n, position of nuclei.
Mentions: Rng3p-GFP3 and Rng3p-YFP3 concentrated in contractile rings from anaphase B through constriction (Fig. 5). CFP-Myo2p joins the contractile ring earlier, beginning with a broad band of small dots before condensing into the contractile ring (Wu et al., 2003). Thus, 21% of full-diameter CFP-Myo2p contractile rings lacked detectable Rng3p-YFP3, whereas both CFP-Myo2p and Rng3p-YFP3 concentrated in all constricting contractile rings (Fig. 5, C–E; note the positions of the nuclei in Fig. 5 D; Video 4, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). The Rng3p-YFP3 signal was weak, so we cannot rule out low concentrations of Rng3-YFP3 in immature contractile rings, but Rng3p lags behind Myo2p incorporation into the ring.

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