Article

Paxillin and Hic-5 Interaction with Vinculin Is Differentially Regulated by Rac1 and RhoA

Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York, United States of America.
PLoS ONE (Impact Factor: 3.53). 05/2012; 7(5):e37990. DOI: 10.1371/journal.pone.0037990
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
116 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cells use integrin receptors to adhere onto surfaces by binding to ligands such as the Arginine-Glycine-Aspartic Acid (RGD) motif. Cancer cells make use of this adhesion process, which has motivated the development of integrin-directed drugs. However, those drugs may exert paradoxical effects on tumor progression, which raises the question of how integrin function is governed in tumor cells on the nanoscale. We have utilized precisely defined and tunable RGD ligand site densities spanning one order of magnitude, i.e. 103 to 1,145 ligand sites/µm2 by using RGD functionalized gold nanoparticle patterns immobilized on glass by block copolymer (micellar) nanolithography. In an αVβ3 integrin-dependent fashion, human melanoma cells spread, formed focal contacts and re-organized cytoskeletal fibers on a physiologically relevant RGD density of 349 sites/µm². Intriguingly, low doses of solute RGD "shifted" the optimal densities of immobilized ligand along with corresponding melanoma cell integrin clusters and cytoskeletal changes towards those typical for "intermediate" ligand presentation. Consequently, melanoma cells were forced into a "permissive" state optimizing interaction with sub-optimal nanostructured biomimetic surfaces, thus providing an explanation for the seemingly paradoxical effects on tumor progression and a potential clue for individualized anti-tumoral therapies.
    ACS Nano 08/2014; 8(9). DOI:10.1021/nn502690b · 12.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Polarized cell migration is essential for normal organism development and is also a critical component of cancer cell invasion and disease progression. Directional cell motility requires the coordination of dynamic cell-extracellular matrix interactions as well as repositioning of the Golgi apparatus, both of which can be controlled by the microtubule (MT) cytoskeleton. In this paper, we have identified a new and conserved role for the focal adhesion scaffold protein paxillin in regulating the posttranslational modification of the MT cytoskeleton through an inhibitory interaction with the α-tubulin deacetylase HDAC6. We also determined that through HDAC6-dependent regulation of the MT cytoskeleton, paxillin regulates both Golgi organelle integrity and polarized cell invasion and migration in both three-dimensional and two-dimensional matrix microenvironments. Importantly, these data reveal a fundamental role for paxillin in coordinating MT acetylation-dependent cell polarization and migration in both normal and transformed cells.
    The Journal of Cell Biology 07/2014; DOI:10.1083/jcb.201403039 · 9.69 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We investigated cross-talk between the membrane-associated, myosin II-regulatory protein supervillin and the actin-regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1-GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLa cells with normal levels of the Rac1-activating protein Trio. No reduction in Rac1-GTP loading was observed when supervillin levels were reduced in Trio-depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio-mediated Rac1 guanine nucleotide exchange (GEF) activity with ITX3 partially blocked the SV4-mediated increase in Rac1-GTP. Both SV4 and SV1 co-localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4-7: SV1-171, which contains N-terminal residues found in both SV1 and SV4 and the SV4-specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4-E345; SV4 amino acids 276 – 669). In addition, SV4-E345 interacted with the homologous sequence in rat kalirin (repeats 4-7, amino acids 531 - 1101). Overexpressed SV1-174 and SV4-E345 affected Rac1-GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771 – 1057, which contain both supervillin-interaction sites, exerted a dominant-negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension. This article is protected by copyright. All rights reserved.
    Cytoskeleton 02/2015; 72(1). DOI:10.1002/cm.21210 · 3.01 Impact Factor