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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: Rgf3 is an essential GEF for Rho1 GTPase in fission yeast.Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1.Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site.

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: Rgf3 is an essential GEF for Rho1 GTPase in fission yeast.Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1.Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site.

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