Structural details and mechanism of filamentous actin organization by the isoforms vinculin and metavinculin
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Structural details and mechanism of filamentous actin organization by the isoforms vinculin and metavinculin

  • Author(s): Janssen, Maria Elisabeth
  • Advisor(s): Hanein, Dorit
  • et al.
Abstract

Vinculin and its splice variant metavinculin are actin binding proteins involved in the organization of actin filaments, which is necessary in cellular processes such as cell migration, division and differentiation. Vinculin is essential in formation of a stable link between the actin cytoskeleton and the cell membrane via proteins such as talin and a-catenin. A lack of vinculin causes cells to adhere less and have a higher motility, while in vivo this results in impaired neuronal development and heart malfunction causing early death. In this dissertation, I characterized the structural details of how vinculin and metavinculin organize actin filaments.

Transmission electron microscopy is the only technique to obtain high resolution structural information on actin-vinculin assemblies, since these are too large to solve with NMR and are non-crystallizable. A near-atomic model generated by electronmicroscopy, computational docking and biochemistry reveals the structural details of the actin-vinculin tail interaction. Furthermore, binding to actin filaments causes a conformational change in the vinculin tail domain which exposes its dimerization site and subsequently induces actin bundling. A combinatorial input of two or more ligands has been proposed to activate vinculin. In the presence of F-actin, full length vinculin is activated by a-catenin's CD3 domain and several talin domains. CD3 turns out to be the most efficient activator. A slightly smaller, constituently active vinculin construct binds F-actin similar to the vinculin tail. In addition, the smaller construct induces formation of comparable actin bundles except that it leads to a larger spacing between filaments owing to the presence of the head domain.

Despite an additional 68aa insert in the metavinculin tail domain, both vinculin isoforms bind actin filaments similarly. Interestingly, biochemical and biophysical techniques indicate that the metavinculin tail does not bundle, but instead severs actin filaments in a dose dependent manner. This severing activity is not affected by a cardiomyopathy related mutation in the metavinculin tail insert. However, this mutation might influence the affinity between the head and tail domains.

These studies have lead to a molecular model for actin organization by vinculin in adhesion sites and describe a new function for the isoform metavinculin.

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