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: 3.23). 05/2012; 7(5):e37990. DOI: 10.1371/journal.pone.0037990
Source: PubMed


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.

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    • "This subversion of mechanosensory pathways provides cancer cells with survival signals in unfavorable conditions. Accordingly , the alteration in expression of proteins such as integrins, FAK, CAS, paxillin (Deakin et al., 2012; Tikhmyanova et al., 2010; Zhao and Guan, 2009) or in the activity of ␤ 1 integrin (Indra and Beningo, 2011) are very common in metastatic cancer cell lines. Biomechanical signaling has recently been recognized as being crucial for proper embryogenesis and development. "
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    ABSTRACT: Physicochemical interactions between the cell and its environment are crucial for morphogenesis, tissue homeostasis, remodeling and pathogenesis. Cells form specialized structures like focal adhesions and podosomes that are responsible for bi-directional information exchange between the cell and its surroundings. Besides their role in the transmission of regulatory signals, these structures are also involved in mechanosensing and mechanotransduction. In recent years, many investigations have been carried out to elucidate the mechanisms and consequences of the mechanosensitivity of cells. In this review we discuss the role of the integrin pathway in cellular mechanosensing, focusing on primary mechanosensors, molecules that respond to mechanical stress by changing their conformation. We propose mechanisms by which p130Cas is involved in this process, and emphasize the importance of mechanosensing in cell physiology and the development of diseases
    European Journal of Cell Biology 10/2014; 93(10-12). DOI:10.1016/j.ejcb.2014.07.002 · 3.83 Impact Factor
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    • "Vinculin is a 116-kDa cytoskeletal protein that is involved in the linkage of integrin adhesion molecules to the actin cytoskeleton [32]. Vinculin interacts with many proteins including talin [33], [34], [35], alpha-actinin [36], F-actin [37], [38], ARP2/3 [39], catenin [40], [41], [42], [43], Paxillin [44], Hic-5 [44], VASP [45] and vinexin [46], [47], [48]. Structurally, vinculin is divided into three main domains: an N-terminal head, a flexible proline-rich hinge (neck) region, and a C-terminal tail domain [49]. "
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    ABSTRACT: Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.
    PLoS ONE 01/2014; 9(1):e87373. DOI:10.1371/journal.pone.0087373 · 3.23 Impact Factor
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    • ". 3D CDMs have been utilized to elucidate mechanisms of cancer cell invasion. Deakin and Turner have shown that the adapter proteins hic-5 and paxillin are crucial for cancer cell attachment or for mediating 3D-MA adhesion dynamics in 3D CDMs, respectively [27] "
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    ABSTRACT: Three-dimensional in vitro extracellular matrix models provide a physiological alternative to regular two-dimensional cell culture, though they lack the full diversity of molecular composition and physical properties of whole-animal systems. Cell-derived matrices are extracellular matrices that are the product of matrix secretion and assembly by cells cultured at high density in vitro. After the removal of the cells that produced the matrix, an assembled matrix scaffold is left that closely mimics native stromal fiber organization and molecular content. Cell-derived matrices have been shown to impart in vivo-like responses to cells cultured in these matrices. In this review, we focus on mechanisms through which the distinct molecular and topographical composition of cell-derived matrices directs cellular behavior, specifically through regulation of cell-matrix adhesions and subsequent contributions to the process of cell migration.
    Experimental Cell Research 06/2013; 319(16). DOI:10.1016/j.yexcr.2013.05.030 · 3.25 Impact Factor
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