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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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Active Rho1 GTPase at the division site is reduced in art1∆ cells. (A–D) Micrographs (A and B) and quantifications (C and D) of active Rho1 using biosensor Pkc1(HR-C2)-mECitrine in wt (JW5593) and art1∆ (JW5877) cells. Cells were grown in YE5S liquid medium for 48 h before imaging. (B) Representative cells quantified in D. (C) The global intensity of the Rho1 biosensor in wt and art1∆ cells. *, p < 0.05 in t test. (D) Rho1 biosensor intensity at the division site during septal formation in wt and art1∆ cells. Septal growth was measured as percentage completed septa over cell width. *, p ≤ 0.05. (E) Localization of the Rho1 biosensor in wt (JW5593) and rgf3-s44 mutant (JW6165). Cells were grown in YE5S + 1.2 M sorbitol medium due to the severe lysis of rgf3-s44. Scale bars: 5 μm.
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Figure 6: Active Rho1 GTPase at the division site is reduced in art1∆ cells. (A–D) Micrographs (A and B) and quantifications (C and D) of active Rho1 using biosensor Pkc1(HR-C2)-mECitrine in wt (JW5593) and art1∆ (JW5877) cells. Cells were grown in YE5S liquid medium for 48 h before imaging. (B) Representative cells quantified in D. (C) The global intensity of the Rho1 biosensor in wt and art1∆ cells. *, p < 0.05 in t test. (D) Rho1 biosensor intensity at the division site during septal formation in wt and art1∆ cells. Septal growth was measured as percentage completed septa over cell width. *, p ≤ 0.05. (E) Localization of the Rho1 biosensor in wt (JW5593) and rgf3-s44 mutant (JW6165). Cells were grown in YE5S + 1.2 M sorbitol medium due to the severe lysis of rgf3-s44. Scale bars: 5 μm.

Mentions: Our data have shown that Art1 interacts with Rgf3 and is important for its localization and protein levels. Rgf3 is a GEF known to activate Rho1 (Tajadura et al., 2004). Thus we hypothesized that art1∆ negatively affected active Rho1 levels at the division site. We used the Rho1 biosensor Pkc1(HR-C2) (Kono et al., 2012) tagged with mECitrine to measure active GTP-Rho1 in wt and art1∆ cells (Figure 6, A–D). Although the global level of active Rho1 was slightly higher in art1∆ cells than in wt cells (Figure 6C), the amount of active Rho1 at the division sites in cells with forming and complete septa was significantly lower in art1∆ cells than in wt cells (Figure 6, A, B, and D). This result was confirmed using an rgf3-s44 mutant (Figure 6E) in which Rho1 activity is expected to be affected, because rgf3-s44 has a mutation in the GEF domain (Wu et al., 2010).


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

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

Active Rho1 GTPase at the division site is reduced in art1∆ cells. (A–D) Micrographs (A and B) and quantifications (C and D) of active Rho1 using biosensor Pkc1(HR-C2)-mECitrine in wt (JW5593) and art1∆ (JW5877) cells. Cells were grown in YE5S liquid medium for 48 h before imaging. (B) Representative cells quantified in D. (C) The global intensity of the Rho1 biosensor in wt and art1∆ cells. *, p < 0.05 in t test. (D) Rho1 biosensor intensity at the division site during septal formation in wt and art1∆ cells. Septal growth was measured as percentage completed septa over cell width. *, p ≤ 0.05. (E) Localization of the Rho1 biosensor in wt (JW5593) and rgf3-s44 mutant (JW6165). Cells were grown in YE5S + 1.2 M sorbitol medium due to the severe lysis of rgf3-s44. Scale bars: 5 μm.
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Related In: Results  -  Collection

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Figure 6: Active Rho1 GTPase at the division site is reduced in art1∆ cells. (A–D) Micrographs (A and B) and quantifications (C and D) of active Rho1 using biosensor Pkc1(HR-C2)-mECitrine in wt (JW5593) and art1∆ (JW5877) cells. Cells were grown in YE5S liquid medium for 48 h before imaging. (B) Representative cells quantified in D. (C) The global intensity of the Rho1 biosensor in wt and art1∆ cells. *, p < 0.05 in t test. (D) Rho1 biosensor intensity at the division site during septal formation in wt and art1∆ cells. Septal growth was measured as percentage completed septa over cell width. *, p ≤ 0.05. (E) Localization of the Rho1 biosensor in wt (JW5593) and rgf3-s44 mutant (JW6165). Cells were grown in YE5S + 1.2 M sorbitol medium due to the severe lysis of rgf3-s44. Scale bars: 5 μm.
Mentions: Our data have shown that Art1 interacts with Rgf3 and is important for its localization and protein levels. Rgf3 is a GEF known to activate Rho1 (Tajadura et al., 2004). Thus we hypothesized that art1∆ negatively affected active Rho1 levels at the division site. We used the Rho1 biosensor Pkc1(HR-C2) (Kono et al., 2012) tagged with mECitrine to measure active GTP-Rho1 in wt and art1∆ cells (Figure 6, A–D). Although the global level of active Rho1 was slightly higher in art1∆ cells than in wt cells (Figure 6C), the amount of active Rho1 at the division sites in cells with forming and complete septa was significantly lower in art1∆ cells than in wt cells (Figure 6, A, B, and D). This result was confirmed using an rgf3-s44 mutant (Figure 6E) in which Rho1 activity is expected to be affected, because rgf3-s44 has a mutation in the GEF domain (Wu et al., 2010).

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