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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: 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.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 mutant twinfilin Twf1-3p is not able to bind actin monomers. (A) The ability of wild-type (•), Twf1-1p (○), Twf1-2p (▴), and Twf1-3p (▵) to sequester actin monomers is demonstrated by a graphical representation of the quantitation the amount of actin present in the supernatant fraction in actin filament sedimentation assays. The actin concentration in the experiment was 4 μM and the twinfilin concentration varied from 0 to 8 μM. Wild-type twinfilin efficiently increases the amount of yeast actin in the supernatant, whereas Twf1-3p is much less efficient in increasing the amount of actin in the supernatant. (B) Wild-type twinfilin and Twf1-3p have similar structure and stability. Far UV CD spectra of wild-type twinfilin (solid line) and Twf1-3p (dotted line) at 20°C are almost identical. Twf1-3p appears to be slightly more stable than wild-type twinfilin, as judged on the basis of melting points that were measured by following α helix distortion by CD signal at 222 nm as the temperature was raised in intervals of 5°.
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fig4: The mutant twinfilin Twf1-3p is not able to bind actin monomers. (A) The ability of wild-type (•), Twf1-1p (○), Twf1-2p (▴), and Twf1-3p (▵) to sequester actin monomers is demonstrated by a graphical representation of the quantitation the amount of actin present in the supernatant fraction in actin filament sedimentation assays. The actin concentration in the experiment was 4 μM and the twinfilin concentration varied from 0 to 8 μM. Wild-type twinfilin efficiently increases the amount of yeast actin in the supernatant, whereas Twf1-3p is much less efficient in increasing the amount of actin in the supernatant. (B) Wild-type twinfilin and Twf1-3p have similar structure and stability. Far UV CD spectra of wild-type twinfilin (solid line) and Twf1-3p (dotted line) at 20°C are almost identical. Twf1-3p appears to be slightly more stable than wild-type twinfilin, as judged on the basis of melting points that were measured by following α helix distortion by CD signal at 222 nm as the temperature was raised in intervals of 5°.

Mentions: Purified wild-type and mutant twinfilins were analyzed for actin monomer interactions by actin filament sedimentation and native gel electrophoresis assays. In actin filament sedimentation assays, we compared the ability of wild-type and mutant twinfilins to shift actin from filaments (pellet) to the monomer pool (supernatant). Wild-type twinfilin efficiently shifts actin from filaments to the monomer pool with the monomer-sequestering activity saturated at 4 μM twinfilin. Twf1-1p, in which only residues in the first ADF-H domain are replaced by alanines, is somewhat less efficient in sequestering actin monomers in this assay. Also, Twf1-2p shows detectable actin monomer sequestering activity, but it is significantly less efficient than Twf1-1p in shifting actin into the monomeric fraction. Twf1-3p, in which residues in both ADF-H domains are mutated, can no longer shift detectable amounts of actin into the supernatant fraction with the protein concentration range probed in this study (Fig. 4 A). The defect in actin monomer interactions was also seen in native gel electrophoresis assays. Whereas wild-type twinfilin forms a stable complex with ADP-actin monomers on native gels, no detectable complex formation between Twf1-3p and ADP-actin monomers could be observed (unpublished data). The far UV CD spectra of purified wild-type and Twf1-3p twinfilins are almost identical to each other, suggesting that the composition of secondary structure elements in these proteins is very similar (Fig. 4 B). Additionally, the mutations in Twf1-3p do not decrease the protein stability because wild-type twinfilin has a Tm value of 55°C, whereas the Tm value of Twf1-3p is 59°C.


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 mutant twinfilin Twf1-3p is not able to bind actin monomers. (A) The ability of wild-type (•), Twf1-1p (○), Twf1-2p (▴), and Twf1-3p (▵) to sequester actin monomers is demonstrated by a graphical representation of the quantitation the amount of actin present in the supernatant fraction in actin filament sedimentation assays. The actin concentration in the experiment was 4 μM and the twinfilin concentration varied from 0 to 8 μM. Wild-type twinfilin efficiently increases the amount of yeast actin in the supernatant, whereas Twf1-3p is much less efficient in increasing the amount of actin in the supernatant. (B) Wild-type twinfilin and Twf1-3p have similar structure and stability. Far UV CD spectra of wild-type twinfilin (solid line) and Twf1-3p (dotted line) at 20°C are almost identical. Twf1-3p appears to be slightly more stable than wild-type twinfilin, as judged on the basis of melting points that were measured by following α helix distortion by CD signal at 222 nm as the temperature was raised in intervals of 5°.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198831&req=5

fig4: The mutant twinfilin Twf1-3p is not able to bind actin monomers. (A) The ability of wild-type (•), Twf1-1p (○), Twf1-2p (▴), and Twf1-3p (▵) to sequester actin monomers is demonstrated by a graphical representation of the quantitation the amount of actin present in the supernatant fraction in actin filament sedimentation assays. The actin concentration in the experiment was 4 μM and the twinfilin concentration varied from 0 to 8 μM. Wild-type twinfilin efficiently increases the amount of yeast actin in the supernatant, whereas Twf1-3p is much less efficient in increasing the amount of actin in the supernatant. (B) Wild-type twinfilin and Twf1-3p have similar structure and stability. Far UV CD spectra of wild-type twinfilin (solid line) and Twf1-3p (dotted line) at 20°C are almost identical. Twf1-3p appears to be slightly more stable than wild-type twinfilin, as judged on the basis of melting points that were measured by following α helix distortion by CD signal at 222 nm as the temperature was raised in intervals of 5°.
Mentions: Purified wild-type and mutant twinfilins were analyzed for actin monomer interactions by actin filament sedimentation and native gel electrophoresis assays. In actin filament sedimentation assays, we compared the ability of wild-type and mutant twinfilins to shift actin from filaments (pellet) to the monomer pool (supernatant). Wild-type twinfilin efficiently shifts actin from filaments to the monomer pool with the monomer-sequestering activity saturated at 4 μM twinfilin. Twf1-1p, in which only residues in the first ADF-H domain are replaced by alanines, is somewhat less efficient in sequestering actin monomers in this assay. Also, Twf1-2p shows detectable actin monomer sequestering activity, but it is significantly less efficient than Twf1-1p in shifting actin into the monomeric fraction. Twf1-3p, in which residues in both ADF-H domains are mutated, can no longer shift detectable amounts of actin into the supernatant fraction with the protein concentration range probed in this study (Fig. 4 A). The defect in actin monomer interactions was also seen in native gel electrophoresis assays. Whereas wild-type twinfilin forms a stable complex with ADP-actin monomers on native gels, no detectable complex formation between Twf1-3p and ADP-actin monomers could be observed (unpublished data). The far UV CD spectra of purified wild-type and Twf1-3p twinfilins are almost identical to each other, suggesting that the composition of secondary structure elements in these proteins is very similar (Fig. 4 B). Additionally, the mutations in Twf1-3p do not decrease the protein stability because wild-type twinfilin has a Tm value of 55°C, whereas the Tm value of Twf1-3p is 59°C.

Bottom Line: 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.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