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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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Art1 plays a role in Rgf3 localization. (A) Art1 mislocalizes Rgf3 to Mid1 cortical nodes in single-plane (near cell surface) images of cells expressing Art1-mECitrine Rgf3-mCFP Mid1-GBP (JW5798). (B and C) Art1 mislocalizes Rgf3 to SPBs with Cdc7 in cells expressing Art1-mECitrine Rgf3-mCFP Cdc7-GBP (JW5799). (B) Single focal-plane images. Red boxes show examples of Rgf3 colocalization with mislocalized Art1. (C) Each image shown in the time course is a rolling average of the sum projection (of three z-slices spaced at 0.4 μm) from three consecutive time points at ∼13 s apart. The cell boundary of all cells (A and B) or the cell at 0 s (C) was marked with dashed lines. Scale bars: 5 μm.
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Figure 5: Art1 plays a role in Rgf3 localization. (A) Art1 mislocalizes Rgf3 to Mid1 cortical nodes in single-plane (near cell surface) images of cells expressing Art1-mECitrine Rgf3-mCFP Mid1-GBP (JW5798). (B and C) Art1 mislocalizes Rgf3 to SPBs with Cdc7 in cells expressing Art1-mECitrine Rgf3-mCFP Cdc7-GBP (JW5799). (B) Single focal-plane images. Red boxes show examples of Rgf3 colocalization with mislocalized Art1. (C) Each image shown in the time course is a rolling average of the sum projection (of three z-slices spaced at 0.4 μm) from three consecutive time points at ∼13 s apart. The cell boundary of all cells (A and B) or the cell at 0 s (C) was marked with dashed lines. Scale bars: 5 μm.

Mentions: In art1∆ cells, Rgf3 localized to the septal region, but its localization to the mature contractile ring was severely affected (Figure 4A), which suggested that Art1 was important for Rgf3 localization to the contractile ring. However, the Rgf3 levels were also significantly reduced in art1∆ cells (Figure 4, A–C). Thus it was hard to know whether the lack of Rgf3 localization was due to the reduction in overall protein levels. To test whether Art1 plays a direct role in localizing Rgf3, we mislocalized Art1 to various cellular structures using the GFP-binding protein (GBP; Figure 5 and Supplemental Figure S6; Rothbauer et al., 2008). GBP binds to the YFP variant mECitrine but not to cyan fluorescent protein (CFP; Rothbauer et al., 2008; Grallert et al., 2013); we therefore tested whether mislocalized Art1-mECitrine recruited Rgf3-mCFP. We mislocalized Art1 to cortical nodes using anillin Mid1-GBP (Figure 5A) and to SPBs using SIN kinase Cdc7-GBP (Figure 5, B and C). Mislocalized Art1 successfully recruited Rgf3-mCFP to cortical nodes and SPBs (Figure 5, A–C). Only a fraction of Art1 was mislocalized; we observed that Art1 still localized to its native localizations, the constricting ring and the septum (Figure 5, B and C, and Supplemental Figure S6), where Mid1 and Cdc7 are normally absent (Sohrmann et al., 1998; Paoletti and Chang, 2000; Mehta and Gould, 2006). Two lines of evidence indicate the mislocalization is specific: 1) No bleed-through was seen from the YFP to the CFP (440 nm) channel (Supplemental Figure S6A, middle panel); and 2) although Rlc1 is more abundant in cells than Rgf3 (Wu and Pollard, 2005), Rlc1 was not recruited by mislocalized Art1 to SPBs (Supplemental Figure S6B). Together these data indicate that Art1 is important for both the localization and protein levels of Rgf3.


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

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

Art1 plays a role in Rgf3 localization. (A) Art1 mislocalizes Rgf3 to Mid1 cortical nodes in single-plane (near cell surface) images of cells expressing Art1-mECitrine Rgf3-mCFP Mid1-GBP (JW5798). (B and C) Art1 mislocalizes Rgf3 to SPBs with Cdc7 in cells expressing Art1-mECitrine Rgf3-mCFP Cdc7-GBP (JW5799). (B) Single focal-plane images. Red boxes show examples of Rgf3 colocalization with mislocalized Art1. (C) Each image shown in the time course is a rolling average of the sum projection (of three z-slices spaced at 0.4 μm) from three consecutive time points at ∼13 s apart. The cell boundary of all cells (A and B) or the cell at 0 s (C) was marked with dashed lines. Scale bars: 5 μm.
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Related In: Results  -  Collection

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Figure 5: Art1 plays a role in Rgf3 localization. (A) Art1 mislocalizes Rgf3 to Mid1 cortical nodes in single-plane (near cell surface) images of cells expressing Art1-mECitrine Rgf3-mCFP Mid1-GBP (JW5798). (B and C) Art1 mislocalizes Rgf3 to SPBs with Cdc7 in cells expressing Art1-mECitrine Rgf3-mCFP Cdc7-GBP (JW5799). (B) Single focal-plane images. Red boxes show examples of Rgf3 colocalization with mislocalized Art1. (C) Each image shown in the time course is a rolling average of the sum projection (of three z-slices spaced at 0.4 μm) from three consecutive time points at ∼13 s apart. The cell boundary of all cells (A and B) or the cell at 0 s (C) was marked with dashed lines. Scale bars: 5 μm.
Mentions: In art1∆ cells, Rgf3 localized to the septal region, but its localization to the mature contractile ring was severely affected (Figure 4A), which suggested that Art1 was important for Rgf3 localization to the contractile ring. However, the Rgf3 levels were also significantly reduced in art1∆ cells (Figure 4, A–C). Thus it was hard to know whether the lack of Rgf3 localization was due to the reduction in overall protein levels. To test whether Art1 plays a direct role in localizing Rgf3, we mislocalized Art1 to various cellular structures using the GFP-binding protein (GBP; Figure 5 and Supplemental Figure S6; Rothbauer et al., 2008). GBP binds to the YFP variant mECitrine but not to cyan fluorescent protein (CFP; Rothbauer et al., 2008; Grallert et al., 2013); we therefore tested whether mislocalized Art1-mECitrine recruited Rgf3-mCFP. We mislocalized Art1 to cortical nodes using anillin Mid1-GBP (Figure 5A) and to SPBs using SIN kinase Cdc7-GBP (Figure 5, B and C). Mislocalized Art1 successfully recruited Rgf3-mCFP to cortical nodes and SPBs (Figure 5, A–C). Only a fraction of Art1 was mislocalized; we observed that Art1 still localized to its native localizations, the constricting ring and the septum (Figure 5, B and C, and Supplemental Figure S6), where Mid1 and Cdc7 are normally absent (Sohrmann et al., 1998; Paoletti and Chang, 2000; Mehta and Gould, 2006). Two lines of evidence indicate the mislocalization is specific: 1) No bleed-through was seen from the YFP to the CFP (440 nm) channel (Supplemental Figure S6A, middle panel); and 2) although Rlc1 is more abundant in cells than Rgf3 (Wu and Pollard, 2005), Rlc1 was not recruited by mislocalized Art1 to SPBs (Supplemental Figure S6B). Together these data indicate that Art1 is important for both the localization and protein levels of Rgf3.

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