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Formin and capping protein together embrace the actin filament in a ménage à trois.

Shekhar S, Kerleau M, Kühn S, Pernier J, Romet-Lemonne G, Jégou A, Carlier MF - Nat Commun (2015)

Bottom Line: This is further confirmed using single-molecule imaging.We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins.Implications in filopodia regulation and barbed-end structural regulation are discussed.

View Article: PubMed Central - PubMed

Affiliation: Cytoskeleton Dynamics and Cell Motility, Department of Biochemistry, Biophysics and Structural Biology, I2BC, CNRS, 91198 Gif-sur-Yvette, France.

ABSTRACT
Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed.

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Related in: MedlinePlus

Structural clashes between CP and formin at the barbed end must cause partial dissociation of each protein in the BFC state.(a) Steric clashes between CP and mDia1-FH2 in the BFC complex. The 167°-twisted F-actin barbed end is depicted as surface representation (4A7N), while the α/β heterodimeric CP30 and the dimeric mDia1-FH2 domain (3O4X) are illustrated in ribbon diagrams. The FH2 domain hemidimers (FH21 and FH22) are shown in the previously defined ‘open' state432 and bind with an amphipathic α-helix (αD (ref. 32), orange, knob region) to the hydrophobic target-binding cleft (TBC) of actin. CP interacts with its amphipathic β-tentacle (βT, yellow) with B1, while the basic α-tentacle (αT) binds to B1 and B2. (b,c) Zoom-in of the clashes. The α-tentacle of CP is able to interact with B1 and B2 of F-actin, while there is a steric clash between CPβ and the post region of FH21 (clash 1). FH21 competes with CP for binding to B1-TBC (clash 2). Since CPβ and αD of FH22 are in close proximity, there might be a minor steric hindrance for simultaneous binding to actin B2 (clash 3). (d) Cartoon depicting complex formation and dissociation of BFC, based on the present work and the structural model presented in Supplementary Fig. 13. Left panel: association of PA to the barbed end is prohibited in BC state and permitted in ‘open' BF state. Right panel: formin binds to BC by association of FH22 to B1 and B2 (top) followed by displacement of β-tentacle by FH21 (bottom). Similarly CP can associate with BF without inserting the β-tentacle in B1-TBC (bottom). For the detailed model, see Supplementary Fig. 13.
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f5: Structural clashes between CP and formin at the barbed end must cause partial dissociation of each protein in the BFC state.(a) Steric clashes between CP and mDia1-FH2 in the BFC complex. The 167°-twisted F-actin barbed end is depicted as surface representation (4A7N), while the α/β heterodimeric CP30 and the dimeric mDia1-FH2 domain (3O4X) are illustrated in ribbon diagrams. The FH2 domain hemidimers (FH21 and FH22) are shown in the previously defined ‘open' state432 and bind with an amphipathic α-helix (αD (ref. 32), orange, knob region) to the hydrophobic target-binding cleft (TBC) of actin. CP interacts with its amphipathic β-tentacle (βT, yellow) with B1, while the basic α-tentacle (αT) binds to B1 and B2. (b,c) Zoom-in of the clashes. The α-tentacle of CP is able to interact with B1 and B2 of F-actin, while there is a steric clash between CPβ and the post region of FH21 (clash 1). FH21 competes with CP for binding to B1-TBC (clash 2). Since CPβ and αD of FH22 are in close proximity, there might be a minor steric hindrance for simultaneous binding to actin B2 (clash 3). (d) Cartoon depicting complex formation and dissociation of BFC, based on the present work and the structural model presented in Supplementary Fig. 13. Left panel: association of PA to the barbed end is prohibited in BC state and permitted in ‘open' BF state. Right panel: formin binds to BC by association of FH22 to B1 and B2 (top) followed by displacement of β-tentacle by FH21 (bottom). Similarly CP can associate with BF without inserting the β-tentacle in B1-TBC (bottom). For the detailed model, see Supplementary Fig. 13.

Mentions: We next examined whether the present data and known relevant structures of CP and the formin FH2 domain dimer2930313233 allow simultaneous binding of CP and formin in the BFC state, consistent with its functional properties (see Methods section). The CP αβ heterodimer binds the two terminal actin subunits (B1 and B2) of actin filament barbed ends in the helical, 167°-twisted configuration of F-actin3031. Recent EM data at 4 Å resolution confirm this structural organization in the CapZ-capped dynactin filament34. In contrast, nonpolymerizable actin subunits are oriented in the FH2-actin crystal structure in a helical 180° pseudo F-actin configuration32, a conformation that presumably prevents the formation of main CP barbed-end contacts. Molecular dynamics studies provided insights into the BF complex in a 167°-twisted filament35. To challenge the simultaneous interaction of formin and CP with the standard helical filament barbed end, the mDia1-FH2 dimer (chains FH21 and FH22) was superimposed in the ‘open' state on the two terminal barbed-end protomers. This ‘open' state has been proposed to be adopted during G-actin association4. Most of the FH2-actin contacts are conserved in the 167°-F-actin context, in particular those made by the FH2 knob region, a main actin-binding element. When this structure is superimposed on the BC complex structure, steric clashes appear between CPβ and the post region of the FH21 protomer. In addition, while a strong steric conflict takes place between the β-tentacle of CP and the knob region of FH21, the main electrostatic interactions of CPα with B1 and B2 are not impaired (Fig. 5a–c).


Formin and capping protein together embrace the actin filament in a ménage à trois.

Shekhar S, Kerleau M, Kühn S, Pernier J, Romet-Lemonne G, Jégou A, Carlier MF - Nat Commun (2015)

Structural clashes between CP and formin at the barbed end must cause partial dissociation of each protein in the BFC state.(a) Steric clashes between CP and mDia1-FH2 in the BFC complex. The 167°-twisted F-actin barbed end is depicted as surface representation (4A7N), while the α/β heterodimeric CP30 and the dimeric mDia1-FH2 domain (3O4X) are illustrated in ribbon diagrams. The FH2 domain hemidimers (FH21 and FH22) are shown in the previously defined ‘open' state432 and bind with an amphipathic α-helix (αD (ref. 32), orange, knob region) to the hydrophobic target-binding cleft (TBC) of actin. CP interacts with its amphipathic β-tentacle (βT, yellow) with B1, while the basic α-tentacle (αT) binds to B1 and B2. (b,c) Zoom-in of the clashes. The α-tentacle of CP is able to interact with B1 and B2 of F-actin, while there is a steric clash between CPβ and the post region of FH21 (clash 1). FH21 competes with CP for binding to B1-TBC (clash 2). Since CPβ and αD of FH22 are in close proximity, there might be a minor steric hindrance for simultaneous binding to actin B2 (clash 3). (d) Cartoon depicting complex formation and dissociation of BFC, based on the present work and the structural model presented in Supplementary Fig. 13. Left panel: association of PA to the barbed end is prohibited in BC state and permitted in ‘open' BF state. Right panel: formin binds to BC by association of FH22 to B1 and B2 (top) followed by displacement of β-tentacle by FH21 (bottom). Similarly CP can associate with BF without inserting the β-tentacle in B1-TBC (bottom). For the detailed model, see Supplementary Fig. 13.
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f5: Structural clashes between CP and formin at the barbed end must cause partial dissociation of each protein in the BFC state.(a) Steric clashes between CP and mDia1-FH2 in the BFC complex. The 167°-twisted F-actin barbed end is depicted as surface representation (4A7N), while the α/β heterodimeric CP30 and the dimeric mDia1-FH2 domain (3O4X) are illustrated in ribbon diagrams. The FH2 domain hemidimers (FH21 and FH22) are shown in the previously defined ‘open' state432 and bind with an amphipathic α-helix (αD (ref. 32), orange, knob region) to the hydrophobic target-binding cleft (TBC) of actin. CP interacts with its amphipathic β-tentacle (βT, yellow) with B1, while the basic α-tentacle (αT) binds to B1 and B2. (b,c) Zoom-in of the clashes. The α-tentacle of CP is able to interact with B1 and B2 of F-actin, while there is a steric clash between CPβ and the post region of FH21 (clash 1). FH21 competes with CP for binding to B1-TBC (clash 2). Since CPβ and αD of FH22 are in close proximity, there might be a minor steric hindrance for simultaneous binding to actin B2 (clash 3). (d) Cartoon depicting complex formation and dissociation of BFC, based on the present work and the structural model presented in Supplementary Fig. 13. Left panel: association of PA to the barbed end is prohibited in BC state and permitted in ‘open' BF state. Right panel: formin binds to BC by association of FH22 to B1 and B2 (top) followed by displacement of β-tentacle by FH21 (bottom). Similarly CP can associate with BF without inserting the β-tentacle in B1-TBC (bottom). For the detailed model, see Supplementary Fig. 13.
Mentions: We next examined whether the present data and known relevant structures of CP and the formin FH2 domain dimer2930313233 allow simultaneous binding of CP and formin in the BFC state, consistent with its functional properties (see Methods section). The CP αβ heterodimer binds the two terminal actin subunits (B1 and B2) of actin filament barbed ends in the helical, 167°-twisted configuration of F-actin3031. Recent EM data at 4 Å resolution confirm this structural organization in the CapZ-capped dynactin filament34. In contrast, nonpolymerizable actin subunits are oriented in the FH2-actin crystal structure in a helical 180° pseudo F-actin configuration32, a conformation that presumably prevents the formation of main CP barbed-end contacts. Molecular dynamics studies provided insights into the BF complex in a 167°-twisted filament35. To challenge the simultaneous interaction of formin and CP with the standard helical filament barbed end, the mDia1-FH2 dimer (chains FH21 and FH22) was superimposed in the ‘open' state on the two terminal barbed-end protomers. This ‘open' state has been proposed to be adopted during G-actin association4. Most of the FH2-actin contacts are conserved in the 167°-F-actin context, in particular those made by the FH2 knob region, a main actin-binding element. When this structure is superimposed on the BC complex structure, steric clashes appear between CPβ and the post region of the FH21 protomer. In addition, while a strong steric conflict takes place between the β-tentacle of CP and the knob region of FH21, the main electrostatic interactions of CPα with B1 and B2 are not impaired (Fig. 5a–c).

Bottom Line: This is further confirmed using single-molecule imaging.We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins.Implications in filopodia regulation and barbed-end structural regulation are discussed.

View Article: PubMed Central - PubMed

Affiliation: Cytoskeleton Dynamics and Cell Motility, Department of Biochemistry, Biophysics and Structural Biology, I2BC, CNRS, 91198 Gif-sur-Yvette, France.

ABSTRACT
Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed.

Show MeSH
Related in: MedlinePlus