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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2010)

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.

  • 2 supplemental videos
Cover page: Structural details and mechanism of filamentous actin organization by the isoforms vinculin and metavinculin
  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2017)
    Cover page: Characterization of pH-dependent structural changes in adeno-associated virus by cryo-electron microscopy and three-dimensional image reconstruction
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2016)

    Unlabelled

    The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.

    Importance

    The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.
      Cover page: Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging
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