Limits...
Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.

Palmgren S, Ojala PJ, Wear MA, Cooper JA, Lappalainen P - J. Cell Biol. (2001)

Bottom Line: Purified twinfilin and capping protein form a complex on native gels.Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2.Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular Biotechnology, Institute of Biotechnology, FIN-00014 University of Helsinki, Helsinki, Finland.

ABSTRACT
Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Show MeSH

Related in: MedlinePlus

A mutant twinfilin Twf1-3p that is no longer able to bind actin monomers does not localize to cortical actin patches. Wild-type twinfilin and Twf1-3p were expressed in twfΔ cells (DDY1436) and twinfilin (A and C) and actin (B and D) were visualized. Wild-type twinfilin (A and B) localizes to cortical actin patches, whereas Twf1-3p (C and D) shows diffuse cytoplasmic localization. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198831&req=5

fig5: A mutant twinfilin Twf1-3p that is no longer able to bind actin monomers does not localize to cortical actin patches. Wild-type twinfilin and Twf1-3p were expressed in twfΔ cells (DDY1436) and twinfilin (A and C) and actin (B and D) were visualized. Wild-type twinfilin (A and B) localizes to cortical actin patches, whereas Twf1-3p (C and D) shows diffuse cytoplasmic localization. Bar, 5 μm.

Mentions: To examine whether the localization of twinfilin to the cortical actin cytoskeleton depends upon its ability to bind actin monomers, we expressed wild-type and Twf1-3p mutant twinfilin in a twfΔ yeast strain under a GPD promoter. Based on our quantitative Western blot assays, both wild-type and mutant twinfilin are expressed in approximately tenfold higher levels with this expression system, as compared with wild-type cells in which twinfilin is expressed under a control of its own promoter (unpublished data). However, the expression levels varied from cell to cell; therefore, we could probe the localization of wild-type and mutant twinfilins in cells where their expression levels were similar to wild-type yeast cells. As shown in Fig. 5, A and B, wild-type twinfilin localizes mainly to the cortical actin patches, whereas Twf1-3p shows diffuse cytoplasmic localization (Fig. 5, C and D). This result was also confirmed by constructing GFP fusion proteins of wild-type and Twf1-3p twinfilins. When expressed in yeast cells, the Twf1p–GFP localized to cortical patch-like structures, whereas Twf1-3p–GFP showed diffuse cytoplasmic localization (unpublished data).


Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.

Palmgren S, Ojala PJ, Wear MA, Cooper JA, Lappalainen P - J. Cell Biol. (2001)

A mutant twinfilin Twf1-3p that is no longer able to bind actin monomers does not localize to cortical actin patches. Wild-type twinfilin and Twf1-3p were expressed in twfΔ cells (DDY1436) and twinfilin (A and C) and actin (B and D) were visualized. Wild-type twinfilin (A and B) localizes to cortical actin patches, whereas Twf1-3p (C and D) shows diffuse cytoplasmic localization. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198831&req=5

fig5: A mutant twinfilin Twf1-3p that is no longer able to bind actin monomers does not localize to cortical actin patches. Wild-type twinfilin and Twf1-3p were expressed in twfΔ cells (DDY1436) and twinfilin (A and C) and actin (B and D) were visualized. Wild-type twinfilin (A and B) localizes to cortical actin patches, whereas Twf1-3p (C and D) shows diffuse cytoplasmic localization. Bar, 5 μm.
Mentions: To examine whether the localization of twinfilin to the cortical actin cytoskeleton depends upon its ability to bind actin monomers, we expressed wild-type and Twf1-3p mutant twinfilin in a twfΔ yeast strain under a GPD promoter. Based on our quantitative Western blot assays, both wild-type and mutant twinfilin are expressed in approximately tenfold higher levels with this expression system, as compared with wild-type cells in which twinfilin is expressed under a control of its own promoter (unpublished data). However, the expression levels varied from cell to cell; therefore, we could probe the localization of wild-type and mutant twinfilins in cells where their expression levels were similar to wild-type yeast cells. As shown in Fig. 5, A and B, wild-type twinfilin localizes mainly to the cortical actin patches, whereas Twf1-3p shows diffuse cytoplasmic localization (Fig. 5, C and D). This result was also confirmed by constructing GFP fusion proteins of wild-type and Twf1-3p twinfilins. When expressed in yeast cells, the Twf1p–GFP localized to cortical patch-like structures, whereas Twf1-3p–GFP showed diffuse cytoplasmic localization (unpublished data).

Bottom Line: Purified twinfilin and capping protein form a complex on native gels.Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2.Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular Biotechnology, Institute of Biotechnology, FIN-00014 University of Helsinki, Helsinki, Finland.

ABSTRACT
Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Show MeSH
Related in: MedlinePlus