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Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis.

Davidson R, Laporte D, Wu JQ - Mol. Biol. Cell (2014)

Bottom Line: Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site.As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells.Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program of Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH 43210 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.

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Localization of Art1 to the contractile ring and the septation site during cytokinesis. (A–C) Micrographs of Art1-mECitrine (JW2674) localization at the contractile ring (arrowheads) and septal disk (arrows). (C) Three-dimensional projection of the cells in B imaged with 0.2-μm z-spacing. 1 grid = 0.95 μm. (D) Time course of Art1-mECitrine (JW2674) shows its appearance, constriction, and septal localization. (E and F) Micrographs (E) and quantification (F) of the arrival of Art1 (JW2694) and Rgf3 (JW2748) at the division site using SPB protein Sad1-mCFP as a cell cycle marker. (G) Art1 locates outside of Myo2 in the contractile ring. Micrographs (left) and line scans of fluorescence intensity at the contractile ring (right) in cells expressing both Art1-mECitrine and mCFP-Myo2 (JW4427). The central focal plane was used. (H) Actin-filament independence of Art1 (JW2674) and Rgf3 (JW1105) localization. Scale bars: 5 μm.
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Figure 2: Localization of Art1 to the contractile ring and the septation site during cytokinesis. (A–C) Micrographs of Art1-mECitrine (JW2674) localization at the contractile ring (arrowheads) and septal disk (arrows). (C) Three-dimensional projection of the cells in B imaged with 0.2-μm z-spacing. 1 grid = 0.95 μm. (D) Time course of Art1-mECitrine (JW2674) shows its appearance, constriction, and septal localization. (E and F) Micrographs (E) and quantification (F) of the arrival of Art1 (JW2694) and Rgf3 (JW2748) at the division site using SPB protein Sad1-mCFP as a cell cycle marker. (G) Art1 locates outside of Myo2 in the contractile ring. Micrographs (left) and line scans of fluorescence intensity at the contractile ring (right) in cells expressing both Art1-mECitrine and mCFP-Myo2 (JW4427). The central focal plane was used. (H) Actin-filament independence of Art1 (JW2674) and Rgf3 (JW1105) localization. Scale bars: 5 μm.

Mentions: To further understand Art1 function during later stages of cytokinesis, we examined its localization using a monomeric enhanced Citrine (mECitrine; yellow fluorescent protein [YFP] variant)-tagged strain. Art1 concentrated to the contractile ring during cytokinesis (Figure 2, A–C, arrowheads). After ring constriction, the Art1 signal spread unevenly onto the septal disk (Figure 2, A–C, arrows). In addition, Art1 also diffused in the cytoplasm at all stages of the cell cycle. A compact contractile ring forms from cytokinetic nodes at the end of anaphase A in wt cells, when the spindle is ∼2.5 μm long (Wu et al., 2003; Lee et al., 2012). Using time-lapse microscopy, we found that Art1 appeared directly in the contractile ring (not in the precursor nodes), increased in fluorescence intensity, constricted, and then spread to the septum (Figure 2D). Art1 appeared in the contractile ring when spindle pole bodies (SPBs) were ∼3 μm apart (Figure 2, E and F), corresponding to the maturing contractile ring in wt cells at early anaphase B (Nabeshima et al., 1998; Mallavarapu et al., 1999; Wu et al., 2003). It remained at the division site until cell separation. Thus the pattern of Art1 localization is consistent with its role in contractile-ring maturation, ring constriction, and septal integrity.


Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis.

Davidson R, Laporte D, Wu JQ - Mol. Biol. Cell (2014)

Localization of Art1 to the contractile ring and the septation site during cytokinesis. (A–C) Micrographs of Art1-mECitrine (JW2674) localization at the contractile ring (arrowheads) and septal disk (arrows). (C) Three-dimensional projection of the cells in B imaged with 0.2-μm z-spacing. 1 grid = 0.95 μm. (D) Time course of Art1-mECitrine (JW2674) shows its appearance, constriction, and septal localization. (E and F) Micrographs (E) and quantification (F) of the arrival of Art1 (JW2694) and Rgf3 (JW2748) at the division site using SPB protein Sad1-mCFP as a cell cycle marker. (G) Art1 locates outside of Myo2 in the contractile ring. Micrographs (left) and line scans of fluorescence intensity at the contractile ring (right) in cells expressing both Art1-mECitrine and mCFP-Myo2 (JW4427). The central focal plane was used. (H) Actin-filament independence of Art1 (JW2674) and Rgf3 (JW1105) localization. Scale bars: 5 μm.
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Related In: Results  -  Collection

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Figure 2: Localization of Art1 to the contractile ring and the septation site during cytokinesis. (A–C) Micrographs of Art1-mECitrine (JW2674) localization at the contractile ring (arrowheads) and septal disk (arrows). (C) Three-dimensional projection of the cells in B imaged with 0.2-μm z-spacing. 1 grid = 0.95 μm. (D) Time course of Art1-mECitrine (JW2674) shows its appearance, constriction, and septal localization. (E and F) Micrographs (E) and quantification (F) of the arrival of Art1 (JW2694) and Rgf3 (JW2748) at the division site using SPB protein Sad1-mCFP as a cell cycle marker. (G) Art1 locates outside of Myo2 in the contractile ring. Micrographs (left) and line scans of fluorescence intensity at the contractile ring (right) in cells expressing both Art1-mECitrine and mCFP-Myo2 (JW4427). The central focal plane was used. (H) Actin-filament independence of Art1 (JW2674) and Rgf3 (JW1105) localization. Scale bars: 5 μm.
Mentions: To further understand Art1 function during later stages of cytokinesis, we examined its localization using a monomeric enhanced Citrine (mECitrine; yellow fluorescent protein [YFP] variant)-tagged strain. Art1 concentrated to the contractile ring during cytokinesis (Figure 2, A–C, arrowheads). After ring constriction, the Art1 signal spread unevenly onto the septal disk (Figure 2, A–C, arrows). In addition, Art1 also diffused in the cytoplasm at all stages of the cell cycle. A compact contractile ring forms from cytokinetic nodes at the end of anaphase A in wt cells, when the spindle is ∼2.5 μm long (Wu et al., 2003; Lee et al., 2012). Using time-lapse microscopy, we found that Art1 appeared directly in the contractile ring (not in the precursor nodes), increased in fluorescence intensity, constricted, and then spread to the septum (Figure 2D). Art1 appeared in the contractile ring when spindle pole bodies (SPBs) were ∼3 μm apart (Figure 2, E and F), corresponding to the maturing contractile ring in wt cells at early anaphase B (Nabeshima et al., 1998; Mallavarapu et al., 1999; Wu et al., 2003). It remained at the division site until cell separation. Thus the pattern of Art1 localization is consistent with its role in contractile-ring maturation, ring constriction, and septal integrity.

Bottom Line: Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site.As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells.Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program of Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH 43210 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.

Show MeSH
Related in: MedlinePlus