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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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Twinfilin interacts with capping protein. (A) Immunoprecipitation of twinfilin with anti-yeast twinfilin antibody was carried out from wild-type (lane 1) and Δtwf1 (lane 2) yeast extracts. The blot detected with an anti-Cap2p antibody shows coimmunoprecipitation of Cap2p (MW ∼33 kD) with twinfilin from wild-type yeast extract. Immunoprecipitation of Cap2p was carried out with anti-Cap2p antibody from wild-type (lane 3) and Δcap1,ΔCap2 (lane 4) strains. The blot detected with an anti-Twf1p antibody shows specific coimmunoprecipitation of twinfilin (MW ∼40 kD) with capping protein. The ∼80-kD band seen in lanes 1 and 2 is a protein that binds unspecifically to protein A-Sepharose beads and cross-reacts with the Cap2p antiserum. (B) The interaction between yeast twinfilin and yeast capping protein was also investigated by native gel electrophoresis. Lane 1, 10 μM twinfilin; lane 2, 10 μM Twf1-3p; lane 3, 5 μM Cap1/2p; lane 4, 10 μM twinfilin + 5 μM Cap1/2p; lane 5, 10 μM Twf1-3p + 5 μM Cap1/2p. The shift in mobility of Cap1/2p in the presence of twinfilin or Twf1-3p indicates a complex formation. (C) Interaction of mouse twinfilin and mouse α1β2 capping protein. Lane 1, 3 μM twinfilin; lane 2, 3 μM capping protein; lane 3, 3 μM twinfilin + 3 μM capping protein. (D) Analysis in a second dimension on a 12% SDS–polyacrylamide gel of the protein components from B (lane 3) shows the presence of twinfilin and both subunits (α1 and β2) of capping protein in the shifted band.
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fig7: Twinfilin interacts with capping protein. (A) Immunoprecipitation of twinfilin with anti-yeast twinfilin antibody was carried out from wild-type (lane 1) and Δtwf1 (lane 2) yeast extracts. The blot detected with an anti-Cap2p antibody shows coimmunoprecipitation of Cap2p (MW ∼33 kD) with twinfilin from wild-type yeast extract. Immunoprecipitation of Cap2p was carried out with anti-Cap2p antibody from wild-type (lane 3) and Δcap1,ΔCap2 (lane 4) strains. The blot detected with an anti-Twf1p antibody shows specific coimmunoprecipitation of twinfilin (MW ∼40 kD) with capping protein. The ∼80-kD band seen in lanes 1 and 2 is a protein that binds unspecifically to protein A-Sepharose beads and cross-reacts with the Cap2p antiserum. (B) The interaction between yeast twinfilin and yeast capping protein was also investigated by native gel electrophoresis. Lane 1, 10 μM twinfilin; lane 2, 10 μM Twf1-3p; lane 3, 5 μM Cap1/2p; lane 4, 10 μM twinfilin + 5 μM Cap1/2p; lane 5, 10 μM Twf1-3p + 5 μM Cap1/2p. The shift in mobility of Cap1/2p in the presence of twinfilin or Twf1-3p indicates a complex formation. (C) Interaction of mouse twinfilin and mouse α1β2 capping protein. Lane 1, 3 μM twinfilin; lane 2, 3 μM capping protein; lane 3, 3 μM twinfilin + 3 μM capping protein. (D) Analysis in a second dimension on a 12% SDS–polyacrylamide gel of the protein components from B (lane 3) shows the presence of twinfilin and both subunits (α1 and β2) of capping protein in the shifted band.

Mentions: An interaction between twinfilin and Cap1/2p was further examined by a coimmunoprecipitation assay. Cap1/2p coimmunoprecipitates with anti-Twf1p antibody in the presence, but not in the absence, of twinfilin (Fig. 7 A, lanes 1 and 2). Similarly, twinfilin coimmunoprecipitates with anti-Cap2p antibody (Fig. 7 A, lanes 3 and 4), suggesting that these proteins interact with each other in vivo. A native PAGE assay was carried out to elucidate whether purified twinfilin and capping protein interact with each other in vitro. Purified Cap1/2p runs as a single band below the migration position of yeast twinfilin on a native gel (Fig. 7 B). However, when mixed with each other before loading on gel, a shift in the mobility of Cap1/2p is observed, suggesting that the two proteins form a complex. Also, Twf1-3p mutant twinfilin interacts with Cap1/2p in this assay (Fig. 7 B). We also studied the interaction of purified mouse twinfilin and mouse capping protein (α1β2) with this assay. Mixing these two proteins with each other prior the loading on a gel results in a formation of an intermediate mobility band between the original migration positions of these proteins (Fig. 7 C). Based on a Coomassie blue–stained second dimension SDS-PAGE, this complex contains a 1:1:1 molar ratio of twinfilin:α1 subunit:β2 subunit (Fig. 7 D).


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)

Twinfilin interacts with capping protein. (A) Immunoprecipitation of twinfilin with anti-yeast twinfilin antibody was carried out from wild-type (lane 1) and Δtwf1 (lane 2) yeast extracts. The blot detected with an anti-Cap2p antibody shows coimmunoprecipitation of Cap2p (MW ∼33 kD) with twinfilin from wild-type yeast extract. Immunoprecipitation of Cap2p was carried out with anti-Cap2p antibody from wild-type (lane 3) and Δcap1,ΔCap2 (lane 4) strains. The blot detected with an anti-Twf1p antibody shows specific coimmunoprecipitation of twinfilin (MW ∼40 kD) with capping protein. The ∼80-kD band seen in lanes 1 and 2 is a protein that binds unspecifically to protein A-Sepharose beads and cross-reacts with the Cap2p antiserum. (B) The interaction between yeast twinfilin and yeast capping protein was also investigated by native gel electrophoresis. Lane 1, 10 μM twinfilin; lane 2, 10 μM Twf1-3p; lane 3, 5 μM Cap1/2p; lane 4, 10 μM twinfilin + 5 μM Cap1/2p; lane 5, 10 μM Twf1-3p + 5 μM Cap1/2p. The shift in mobility of Cap1/2p in the presence of twinfilin or Twf1-3p indicates a complex formation. (C) Interaction of mouse twinfilin and mouse α1β2 capping protein. Lane 1, 3 μM twinfilin; lane 2, 3 μM capping protein; lane 3, 3 μM twinfilin + 3 μM capping protein. (D) Analysis in a second dimension on a 12% SDS–polyacrylamide gel of the protein components from B (lane 3) shows the presence of twinfilin and both subunits (α1 and β2) of capping protein in the shifted band.
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fig7: Twinfilin interacts with capping protein. (A) Immunoprecipitation of twinfilin with anti-yeast twinfilin antibody was carried out from wild-type (lane 1) and Δtwf1 (lane 2) yeast extracts. The blot detected with an anti-Cap2p antibody shows coimmunoprecipitation of Cap2p (MW ∼33 kD) with twinfilin from wild-type yeast extract. Immunoprecipitation of Cap2p was carried out with anti-Cap2p antibody from wild-type (lane 3) and Δcap1,ΔCap2 (lane 4) strains. The blot detected with an anti-Twf1p antibody shows specific coimmunoprecipitation of twinfilin (MW ∼40 kD) with capping protein. The ∼80-kD band seen in lanes 1 and 2 is a protein that binds unspecifically to protein A-Sepharose beads and cross-reacts with the Cap2p antiserum. (B) The interaction between yeast twinfilin and yeast capping protein was also investigated by native gel electrophoresis. Lane 1, 10 μM twinfilin; lane 2, 10 μM Twf1-3p; lane 3, 5 μM Cap1/2p; lane 4, 10 μM twinfilin + 5 μM Cap1/2p; lane 5, 10 μM Twf1-3p + 5 μM Cap1/2p. The shift in mobility of Cap1/2p in the presence of twinfilin or Twf1-3p indicates a complex formation. (C) Interaction of mouse twinfilin and mouse α1β2 capping protein. Lane 1, 3 μM twinfilin; lane 2, 3 μM capping protein; lane 3, 3 μM twinfilin + 3 μM capping protein. (D) Analysis in a second dimension on a 12% SDS–polyacrylamide gel of the protein components from B (lane 3) shows the presence of twinfilin and both subunits (α1 and β2) of capping protein in the shifted band.
Mentions: An interaction between twinfilin and Cap1/2p was further examined by a coimmunoprecipitation assay. Cap1/2p coimmunoprecipitates with anti-Twf1p antibody in the presence, but not in the absence, of twinfilin (Fig. 7 A, lanes 1 and 2). Similarly, twinfilin coimmunoprecipitates with anti-Cap2p antibody (Fig. 7 A, lanes 3 and 4), suggesting that these proteins interact with each other in vivo. A native PAGE assay was carried out to elucidate whether purified twinfilin and capping protein interact with each other in vitro. Purified Cap1/2p runs as a single band below the migration position of yeast twinfilin on a native gel (Fig. 7 B). However, when mixed with each other before loading on gel, a shift in the mobility of Cap1/2p is observed, suggesting that the two proteins form a complex. Also, Twf1-3p mutant twinfilin interacts with Cap1/2p in this assay (Fig. 7 B). We also studied the interaction of purified mouse twinfilin and mouse capping protein (α1β2) with this assay. Mixing these two proteins with each other prior the loading on a gel results in a formation of an intermediate mobility band between the original migration positions of these proteins (Fig. 7 C). Based on a Coomassie blue–stained second dimension SDS-PAGE, this complex contains a 1:1:1 molar ratio of twinfilin:α1 subunit:β2 subunit (Fig. 7 D).

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