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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.

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The presence of intact capping protein, Cap1/2p, is required for localization of twinfilin to cortical actin patches. The localization of twinfilin (A, C, E and G) and actin (B, D, F and H) was examined in wild-type (A and B), cap1Δ(C and D), cap2Δ (E and F), and cof1-22 (G and H) yeast strains. Twinfilin colocalizes with the cortical actin patches in wild-type and cof1-22 strains, whereas twinfilin shows diffuse cytoplasmic localization in cap1Δ and cap2Δ strains. Bar, 5 μm.
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fig6: The presence of intact capping protein, Cap1/2p, is required for localization of twinfilin to cortical actin patches. The localization of twinfilin (A, C, E and G) and actin (B, D, F and H) was examined in wild-type (A and B), cap1Δ(C and D), cap2Δ (E and F), and cof1-22 (G and H) yeast strains. Twinfilin colocalizes with the cortical actin patches in wild-type and cof1-22 strains, whereas twinfilin shows diffuse cytoplasmic localization in cap1Δ and cap2Δ strains. Bar, 5 μm.

Mentions: Two other yeast strains in which twinfilin showed an abnormal localization are cap1Δ and cap2Δ. These carry deletions of the α and β subunits of actin filament barbed-end capping protein, Cap1/2p. In these cells, twinfilin localized diffusely in the cytoplasm, and stronger staining was only occasionally observed at and/or near cortical actin patches (Fig. 6, C–F). Whereas virtually all actin patches were positive for twinfilin staining in the wild-type parent strain, detectable patch-like twinfilin staining could be observed in <3% of the actin patches in the cap2Δ strain. Based on a quantitative Western blot analysis, the cap2Δ cells have the same level of twinfilin as wild-type cells, demonstrating that the lack of twinfilin localization to the cortical actin patches in the cap2Δ strain is not a result of decreased twinfilin levels (unpublished data). The defect in actin patch localization in the cap2Δ strain is also specific for twinfilin because other actin patch components, such as cofilin and Abp1p, localize normally to cortical actin patches in the cap2Δ strain (unpublished data). These results show that Cap1/2p is required for efficient localization of twinfilin to actin patches. Twinfilin also localizes to the cortical actin patches in the cof1-22 strain (Fig. 6, G and H). In this yeast strain, the actin monomer pool is depleted due to a mutation in the actin filament depolymerizing protein cofilin (Lappalainen and Drubin, 1997). Therefore, it is unlikely that the localization defect observed in cap1Δ and cap2Δ strains results from a decrease in the cytoplasmic actin monomer pool in the absence of Cap1/2p.


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)

The presence of intact capping protein, Cap1/2p, is required for localization of twinfilin to cortical actin patches. The localization of twinfilin (A, C, E and G) and actin (B, D, F and H) was examined in wild-type (A and B), cap1Δ(C and D), cap2Δ (E and F), and cof1-22 (G and H) yeast strains. Twinfilin colocalizes with the cortical actin patches in wild-type and cof1-22 strains, whereas twinfilin shows diffuse cytoplasmic localization in cap1Δ and cap2Δ strains. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The presence of intact capping protein, Cap1/2p, is required for localization of twinfilin to cortical actin patches. The localization of twinfilin (A, C, E and G) and actin (B, D, F and H) was examined in wild-type (A and B), cap1Δ(C and D), cap2Δ (E and F), and cof1-22 (G and H) yeast strains. Twinfilin colocalizes with the cortical actin patches in wild-type and cof1-22 strains, whereas twinfilin shows diffuse cytoplasmic localization in cap1Δ and cap2Δ strains. Bar, 5 μm.
Mentions: Two other yeast strains in which twinfilin showed an abnormal localization are cap1Δ and cap2Δ. These carry deletions of the α and β subunits of actin filament barbed-end capping protein, Cap1/2p. In these cells, twinfilin localized diffusely in the cytoplasm, and stronger staining was only occasionally observed at and/or near cortical actin patches (Fig. 6, C–F). Whereas virtually all actin patches were positive for twinfilin staining in the wild-type parent strain, detectable patch-like twinfilin staining could be observed in <3% of the actin patches in the cap2Δ strain. Based on a quantitative Western blot analysis, the cap2Δ cells have the same level of twinfilin as wild-type cells, demonstrating that the lack of twinfilin localization to the cortical actin patches in the cap2Δ strain is not a result of decreased twinfilin levels (unpublished data). The defect in actin patch localization in the cap2Δ strain is also specific for twinfilin because other actin patch components, such as cofilin and Abp1p, localize normally to cortical actin patches in the cap2Δ strain (unpublished data). These results show that Cap1/2p is required for efficient localization of twinfilin to actin patches. Twinfilin also localizes to the cortical actin patches in the cof1-22 strain (Fig. 6, G and H). In this yeast strain, the actin monomer pool is depleted due to a mutation in the actin filament depolymerizing protein cofilin (Lappalainen and Drubin, 1997). Therefore, it is unlikely that the localization defect observed in cap1Δ and cap2Δ strains results from a decrease in the cytoplasmic actin monomer pool in the absence of Cap1/2p.

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