33 research outputs found
Paxillin and Hic-5 Interaction with Vinculin Is Differentially Regulated by Rac1 and RhoA
Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases Rac1 and RhoA are indispensable for cell migration as they regulate cell protrusion, cell-extracellular matrix (ECM) interactions and force transduction. However, the consequences of their activity at a molecular level within the cell remain undetermined. Using a combination of FRET, FRAP and biochemical analyses we show that the interactions between the focal adhesion proteins vinculin and paxillin, as well as the closely related family member Hic-5 are spatially and reciprocally regulated by the activity of Rac1 and RhoA. Vinculin in its active conformation interacts with either paxillin or Hic-5 in adhesions in response to Rac1 and RhoA activation respectively, while inactive vinculin interacts with paxillin in the membrane following Rac1 inhibition. Additionally, Rac1 specifically regulates the dynamics of paxillin as well as its binding partner and F-actin interacting protein actopaxin (α-parvin) in adhesions. Furthermore, FRET analysis of protein:protein interactions within cell adhesions formed in 3D matrices revealed that, in contrast to 2D systems vinculin interacts preferentially with Hic-5. This study provides new insight into the complexity of cell-ECM adhesions in both 2D and 3D matrices by providing the first description of RhoGTPase-coordinated protein:protein interactions in a cellular microenvironment. These data identify discrete roles for paxillin and Hic-5 in Rac1 and RhoA-dependent cell adhesion formation and maturation; processes essential for productive cell migration
Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior
α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain
Hypoxia alters the recruitment of tropomyosins into the actin stress fibres of neuroblastoma cells
Complications Associated with the Use of Vacuum Constriction Devices for Erectile Dysfunction in the Spinal Cord Injured Population
Vinculin association with actin cytoskeleton is necessary for stiffness-dependent regulation of vinculin behavior
Surgical Complications Related to Insertion of Penile Prostheses with Emphasis on Infection and Cost
Phosphorylation at Y1065 in Vinculin Mediates Actin Bundling, Cell Spreading, and Mechanical Responses to Force
Vinculin
is an essential structural adaptor protein that localizes
to sites of adhesion and is involved in a number of cell processes
including adhesion, spreading, motility, force transduction, and cell
survival. The C-terminal vinculin tail domain (Vt) contains the necessary
structural components to bind and cross-link actin filaments. Actin
binding to Vt induces a conformational change that promotes dimerization
through the C-terminal hairpin of Vt and enables actin filament cross-linking.
Here we show that Src phosphorylation of Y1065 within the C-terminal
hairpin regulates Vt-mediated actin bundling and provide a detailed
characterization of Y1065 mutations. Furthermore, we show that phosphorylation
at Y1065 plays a role in cell spreading and the response to the application
of mechanical force