Limits...
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.

Show MeSH

Related in: MedlinePlus

The BFC state splits into CP-capped (BC) and formin mDia1-bound (BF) states.(a) Schematic representation of the experimental set-up #2. Fluorescent actin filaments are initiated from formins anchored on the coverslip. The filaments are then sequentially exposed to flows containing PA (non-fluorescent) and CP as indicated. (b) Kymographs of two formin-anchored (indicated by the yellow dot) filaments switching from the rapidly elongating state (BF) to the pausing state (BFC) on binding CP, followed by the transition to either BF (top) or BC (bottom). Rapidly elongating filaments were initiated by exposing formins to the fluorescent actin and profilin. Filaments were then exposed to a solution containing PA with non-fluorescent actin and 100 nM CP for 30 s. On removal of CP from the flow and introduction of non-fluorescent PA, filaments either resume fast elongation (BFC→BF) or detach (BFC→BC). Elongation by formin in non-fluorescent actin gives the appearance of ‘fluorescent segment' moving further away. (c) Fraction of BFC filaments (black symbols; n=76 filaments) undergoing dissociation into either BC (filaments released in the flow, blue squares) or BF (filaments resuming fast growth, red circles). For comparison, the time course of BF produced from BFC in set-up #1 (Fig. 1) is plotted (open red circles). The solid lines are the exponential fits. The data were fitted with an exponential process (continuous line) consistent with a rate constant kobs=k′−F+k′−C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660058&req=5

f2: The BFC state splits into CP-capped (BC) and formin mDia1-bound (BF) states.(a) Schematic representation of the experimental set-up #2. Fluorescent actin filaments are initiated from formins anchored on the coverslip. The filaments are then sequentially exposed to flows containing PA (non-fluorescent) and CP as indicated. (b) Kymographs of two formin-anchored (indicated by the yellow dot) filaments switching from the rapidly elongating state (BF) to the pausing state (BFC) on binding CP, followed by the transition to either BF (top) or BC (bottom). Rapidly elongating filaments were initiated by exposing formins to the fluorescent actin and profilin. Filaments were then exposed to a solution containing PA with non-fluorescent actin and 100 nM CP for 30 s. On removal of CP from the flow and introduction of non-fluorescent PA, filaments either resume fast elongation (BFC→BF) or detach (BFC→BC). Elongation by formin in non-fluorescent actin gives the appearance of ‘fluorescent segment' moving further away. (c) Fraction of BFC filaments (black symbols; n=76 filaments) undergoing dissociation into either BC (filaments released in the flow, blue squares) or BF (filaments resuming fast growth, red circles). For comparison, the time course of BF produced from BFC in set-up #1 (Fig. 1) is plotted (open red circles). The solid lines are the exponential fits. The data were fitted with an exponential process (continuous line) consistent with a rate constant kobs=k′−F+k′−C.

Mentions: Formation of the ternary BFC complex was tested using set-up #2 (Fig. 2a), which may mimic the condition in which the formins are activated and immobilized at a membrane. In contrast with set-up #1, set-up #2 allows us to clearly distinguish the capped BFC state (attached filament) from the capped BC state (detached filament, lost in the flow) and the BF state (resumption of fast elongation).


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)

The BFC state splits into CP-capped (BC) and formin mDia1-bound (BF) states.(a) Schematic representation of the experimental set-up #2. Fluorescent actin filaments are initiated from formins anchored on the coverslip. The filaments are then sequentially exposed to flows containing PA (non-fluorescent) and CP as indicated. (b) Kymographs of two formin-anchored (indicated by the yellow dot) filaments switching from the rapidly elongating state (BF) to the pausing state (BFC) on binding CP, followed by the transition to either BF (top) or BC (bottom). Rapidly elongating filaments were initiated by exposing formins to the fluorescent actin and profilin. Filaments were then exposed to a solution containing PA with non-fluorescent actin and 100 nM CP for 30 s. On removal of CP from the flow and introduction of non-fluorescent PA, filaments either resume fast elongation (BFC→BF) or detach (BFC→BC). Elongation by formin in non-fluorescent actin gives the appearance of ‘fluorescent segment' moving further away. (c) Fraction of BFC filaments (black symbols; n=76 filaments) undergoing dissociation into either BC (filaments released in the flow, blue squares) or BF (filaments resuming fast growth, red circles). For comparison, the time course of BF produced from BFC in set-up #1 (Fig. 1) is plotted (open red circles). The solid lines are the exponential fits. The data were fitted with an exponential process (continuous line) consistent with a rate constant kobs=k′−F+k′−C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660058&req=5

f2: The BFC state splits into CP-capped (BC) and formin mDia1-bound (BF) states.(a) Schematic representation of the experimental set-up #2. Fluorescent actin filaments are initiated from formins anchored on the coverslip. The filaments are then sequentially exposed to flows containing PA (non-fluorescent) and CP as indicated. (b) Kymographs of two formin-anchored (indicated by the yellow dot) filaments switching from the rapidly elongating state (BF) to the pausing state (BFC) on binding CP, followed by the transition to either BF (top) or BC (bottom). Rapidly elongating filaments were initiated by exposing formins to the fluorescent actin and profilin. Filaments were then exposed to a solution containing PA with non-fluorescent actin and 100 nM CP for 30 s. On removal of CP from the flow and introduction of non-fluorescent PA, filaments either resume fast elongation (BFC→BF) or detach (BFC→BC). Elongation by formin in non-fluorescent actin gives the appearance of ‘fluorescent segment' moving further away. (c) Fraction of BFC filaments (black symbols; n=76 filaments) undergoing dissociation into either BC (filaments released in the flow, blue squares) or BF (filaments resuming fast growth, red circles). For comparison, the time course of BF produced from BFC in set-up #1 (Fig. 1) is plotted (open red circles). The solid lines are the exponential fits. The data were fitted with an exponential process (continuous line) consistent with a rate constant kobs=k′−F+k′−C.
Mentions: Formation of the ternary BFC complex was tested using set-up #2 (Fig. 2a), which may mimic the condition in which the formins are activated and immobilized at a membrane. In contrast with set-up #1, set-up #2 allows us to clearly distinguish the capped BFC state (attached filament) from the capped BC state (detached filament, lost in the flow) and the BF state (resumption of fast elongation).

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