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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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Formin mDia1 binds to CP-capped filaments and rapidly uncaps via a transient BCF state.(a) Pyrene actin polymerization assay demonstrates uncapping of capped barbed ends (BC), by formin. Capped filaments (5 μM F-actin, 2% pyrenyl labelled and 5 nM CP) were diluted 50-fold in F-buffer containing 2 μM G-actin (2% pyrenyl labelled) and 6 μM profilin and the following additions: none (black), 2 nM formin (blue) and 4 μM CIN85 (magenta). Red curve is a filament nucleation control (2 μM actin, 6 μM profilin and 2 nM formin) in the absence of CP-capped filaments. Note that a small percentage of non-capped filaments are responsible for the non-zero initial rate in the black (free barbed ends) and blue (formin-bound barbed ends) curves. Dead time is about 20 s. (b) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin mDia1. Filament was elongated from anchored spectrin–actin seeds in the presence of PA, then exposed to 20 nM CP and PA for 1 min (B+C→BC). The capped filament is later exposed to 40 nM formin in the absence of PA for 40 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). (c) Fraction of filaments (in experiment described in b) that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 10 nM (black, n=91 filaments), 20 nM (red, n=50 filaments) and 40 nM (blue, n=69 filaments) formin for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 9 for details. (d) The maximum fraction of BF filaments (plateau values of curves such as shown in c) as a function of formin concentration [F] times the exposure duration (Texpo). The solid line is an exponential fit corresponding to equation (3). Inset: kobs=k′−C+k′−F is independent of the experimental condition. Horizontal lines represent the average (blue) plus or minus the s.d. (grey). Error bars: s.e.m.
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f3: Formin mDia1 binds to CP-capped filaments and rapidly uncaps via a transient BCF state.(a) Pyrene actin polymerization assay demonstrates uncapping of capped barbed ends (BC), by formin. Capped filaments (5 μM F-actin, 2% pyrenyl labelled and 5 nM CP) were diluted 50-fold in F-buffer containing 2 μM G-actin (2% pyrenyl labelled) and 6 μM profilin and the following additions: none (black), 2 nM formin (blue) and 4 μM CIN85 (magenta). Red curve is a filament nucleation control (2 μM actin, 6 μM profilin and 2 nM formin) in the absence of CP-capped filaments. Note that a small percentage of non-capped filaments are responsible for the non-zero initial rate in the black (free barbed ends) and blue (formin-bound barbed ends) curves. Dead time is about 20 s. (b) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin mDia1. Filament was elongated from anchored spectrin–actin seeds in the presence of PA, then exposed to 20 nM CP and PA for 1 min (B+C→BC). The capped filament is later exposed to 40 nM formin in the absence of PA for 40 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). (c) Fraction of filaments (in experiment described in b) that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 10 nM (black, n=91 filaments), 20 nM (red, n=50 filaments) and 40 nM (blue, n=69 filaments) formin for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 9 for details. (d) The maximum fraction of BF filaments (plateau values of curves such as shown in c) as a function of formin concentration [F] times the exposure duration (Texpo). The solid line is an exponential fit corresponding to equation (3). Inset: kobs=k′−C+k′−F is independent of the experimental condition. Horizontal lines represent the average (blue) plus or minus the s.d. (grey). Error bars: s.e.m.

Mentions: We then investigated if mDia1 could also associate to CP-capped barbed ends to form a ternary BCF complex and if BCF is biochemically equivalent to BFC. This was first tested in bulk solution assays by diluting (50-fold) Pyrenyl-labelled CP-capped filaments in F-buffer containing PA (Fig. 3a). The dilution decreased the concentration of free CP to <100 pM, thus making the re-binding of CP to barbed ends following its dissociation negligible. Expectedly, capped filaments failed to grow significantly in the presence of 2 μM PA. However, when mDia1 was added to the mix of capped filaments and PA, processive actin polymerization occured and caused an acceleration, in conditions where mDia1 nucleation is negligible (Fig. 3a). The acceleration matches the time course of CP displacement by formin. No such acceleration was observed when rapid uncapping was elicited by CIN85, a conventional uncapper of the CapZIP family2627, thus this control curve bends downward in a conventional first-order growth process (Fig. 3a). This result provides qualitative indication that mDia1 binds capped barbed ends and uncaps CP, promoting fast growth, before CP has time to dissociate spontaneously (Supplementary Fig. 5b).


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)

Formin mDia1 binds to CP-capped filaments and rapidly uncaps via a transient BCF state.(a) Pyrene actin polymerization assay demonstrates uncapping of capped barbed ends (BC), by formin. Capped filaments (5 μM F-actin, 2% pyrenyl labelled and 5 nM CP) were diluted 50-fold in F-buffer containing 2 μM G-actin (2% pyrenyl labelled) and 6 μM profilin and the following additions: none (black), 2 nM formin (blue) and 4 μM CIN85 (magenta). Red curve is a filament nucleation control (2 μM actin, 6 μM profilin and 2 nM formin) in the absence of CP-capped filaments. Note that a small percentage of non-capped filaments are responsible for the non-zero initial rate in the black (free barbed ends) and blue (formin-bound barbed ends) curves. Dead time is about 20 s. (b) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin mDia1. Filament was elongated from anchored spectrin–actin seeds in the presence of PA, then exposed to 20 nM CP and PA for 1 min (B+C→BC). The capped filament is later exposed to 40 nM formin in the absence of PA for 40 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). (c) Fraction of filaments (in experiment described in b) that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 10 nM (black, n=91 filaments), 20 nM (red, n=50 filaments) and 40 nM (blue, n=69 filaments) formin for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 9 for details. (d) The maximum fraction of BF filaments (plateau values of curves such as shown in c) as a function of formin concentration [F] times the exposure duration (Texpo). The solid line is an exponential fit corresponding to equation (3). Inset: kobs=k′−C+k′−F is independent of the experimental condition. Horizontal lines represent the average (blue) plus or minus the s.d. (grey). Error bars: s.e.m.
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f3: Formin mDia1 binds to CP-capped filaments and rapidly uncaps via a transient BCF state.(a) Pyrene actin polymerization assay demonstrates uncapping of capped barbed ends (BC), by formin. Capped filaments (5 μM F-actin, 2% pyrenyl labelled and 5 nM CP) were diluted 50-fold in F-buffer containing 2 μM G-actin (2% pyrenyl labelled) and 6 μM profilin and the following additions: none (black), 2 nM formin (blue) and 4 μM CIN85 (magenta). Red curve is a filament nucleation control (2 μM actin, 6 μM profilin and 2 nM formin) in the absence of CP-capped filaments. Note that a small percentage of non-capped filaments are responsible for the non-zero initial rate in the black (free barbed ends) and blue (formin-bound barbed ends) curves. Dead time is about 20 s. (b) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin mDia1. Filament was elongated from anchored spectrin–actin seeds in the presence of PA, then exposed to 20 nM CP and PA for 1 min (B+C→BC). The capped filament is later exposed to 40 nM formin in the absence of PA for 40 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). (c) Fraction of filaments (in experiment described in b) that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 10 nM (black, n=91 filaments), 20 nM (red, n=50 filaments) and 40 nM (blue, n=69 filaments) formin for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 9 for details. (d) The maximum fraction of BF filaments (plateau values of curves such as shown in c) as a function of formin concentration [F] times the exposure duration (Texpo). The solid line is an exponential fit corresponding to equation (3). Inset: kobs=k′−C+k′−F is independent of the experimental condition. Horizontal lines represent the average (blue) plus or minus the s.d. (grey). Error bars: s.e.m.
Mentions: We then investigated if mDia1 could also associate to CP-capped barbed ends to form a ternary BCF complex and if BCF is biochemically equivalent to BFC. This was first tested in bulk solution assays by diluting (50-fold) Pyrenyl-labelled CP-capped filaments in F-buffer containing PA (Fig. 3a). The dilution decreased the concentration of free CP to <100 pM, thus making the re-binding of CP to barbed ends following its dissociation negligible. Expectedly, capped filaments failed to grow significantly in the presence of 2 μM PA. However, when mDia1 was added to the mix of capped filaments and PA, processive actin polymerization occured and caused an acceleration, in conditions where mDia1 nucleation is negligible (Fig. 3a). The acceleration matches the time course of CP displacement by formin. No such acceleration was observed when rapid uncapping was elicited by CIN85, a conventional uncapper of the CapZIP family2627, thus this control curve bends downward in a conventional first-order growth process (Fig. 3a). This result provides qualitative indication that mDia1 binds capped barbed ends and uncaps CP, promoting fast growth, before CP has time to dissociate spontaneously (Supplementary Fig. 5b).

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