Abstract
The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.
Highlights
Vinculin is a large protein of 1066 amino acids made of a N-terminal globular head (Vh)3 comprising subdomains D1–D4, followed by a central polyproline-rich linker and a C-terminal tail (Vt) [1]
Because IpaA unmasks the F-actin-binding domain located in the tail, by disrupting the autoinhibitory interaction, we hypothesized that the capping activity of the IpaA-vinculin complex is located in the tail domain of vinculin
Our data demonstrate that the effect of Vt observed in Fig. 1A corresponds to an inhibition of actin assembly and rule out the quenching of pyrenyl-actin fluorescence by Vt recently proposed by others [34]
Summary
Vinculin is a large protein of 1066 amino acids made of a N-terminal globular head (Vh) comprising subdomains D1–D4, followed by a central polyproline-rich linker and a C-terminal tail (Vt) [1] (see Fig. 4A). It is clear that the high affinity binding of IpaA to the D1 head subdomain is sufficient to activate vinculin and induce F-actin binding [22,23,24], the ability of the cellular D1-binding proteins (talin and ␣-actinin) to fully activate vinculin is controversial The study of the vinculin-binding protein IpaA, from the bacteria Shigella, showed that the complex IpaA-vinculin partially inhibits the elongation of actin filament barbed ends [24]. Whether this activity is an intrinsic property of vinculin that is unmasked by IpaA is not known. We show that vinculin and the formin mDia, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends
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