Search

Integrin-mediated focal adhesions (FAs) are large, multi-protein complexes that link the actin cytoskeleton to the extracellular matrix and take part in adhesion-mediated signaling. These adhesions are highly complex and diverse at the molecular level; thus, assigning particular structural or signaling functions to specific components is highly challenging. Here, we combined functional, structural and biophysical approaches to assess the role of a major FA component, namely, integrin-linked kinase (ILK), in adhesion formation. We show here that ILK plays a key role in the formation of focal complexes, early forms of integrin adhesions, and confirm its involvement in the assembly of fibronectin-bound fibrillar adhesions. Examination of ILK-null fibroblasts by cryo-electron tomography pointed to major structural changes in their FAs, manifested as disarray of the associated actin filaments and an increase in the packing density of FA-related particles. Interestingly, adhesion of the mutant cells to the substrate required a higher ligand density than in control cells. These data indicate that ILK has a key role in integrin adhesion assembly and sub-structure, and in the regulation of the FA-associated cytoskeleton. [ABSTRACT FROM AUTHOR]

Mechanical forces are central to developmental, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is ∼2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently. [ABSTRACT FROM AUTHOR]

Integrin-linked kinase (ILK) links integrins to the actin cytoskeleton and is believed to phosphorylate several target proteins. We report that a keratinocyte-restricted deletion of the ILK gene leads to epidermal defects and hair loss. ILK-deficient epidermal keratinocytes exhibited a pronounced integrin-mediated adhesion defect leading to epidermal detachment and blister formation, disruption of the epidermal-dermal basement membrane, and the translocation of proliferating, integrin-expressing keratinocytes to suprabasal epidermal cell layers. [ABSTRACT FROM AUTHOR]

Discusses the dependence of cell motility and tissue invasion on the interaction of the cell with its surrounding environment, such as with neighboring cells, the extracellular matrix and even inorganic bone components. Importance of the focal adhesions to study cell-matrix interactions; Differentiation of cell types; Capability of focal adhesions to mediate the stable attachment of cells to the underlying substratum and anchor the actin cytoskeleton.

Integrin-linked kinase (ILK) and cytoplasmic adaptors of the PINCH and parvin families form a ternary complex, termed IPP, that localizes to integrin adhesions. We show here that deletion of the genes encoding ILK or PINCH1 similarly blocks maturation of focal adhesions to tensin-rich and phosphotyrosine-poor fibrillar adhesions (FBs) by downregulating expression or recruitment of tensin and destabilizing α5β1-integrin-cytoskeleton linkages. As IPP components are interdependent for integrin targeting and protein stability, functional dissection of the complex was achieved by fusing ILK, PINCH, parvin or their individual motifs to the cytoplasmic tail of β3 integrin, normally excluded from FBs. Using this novel gain-of-function approach, we demonstrated that expression of the C-terminal kinase domain of ILK can restore tensin recruitment and prompt focal-adhesion maturation in IPP-null cells. Debilitating mutations in the paxillin- or ATP-binding sites of ILK, together with α-parvin silencing, revealed a determinant role for ILK-parvin association, but not for direct paxillin binding, in this function. We propose a model in which the C-terminal domain of ILK promotes integrin sorting by reinforcing α5β1-integrin-actin linkage and controls force transmission by targeting tensin to maturing adhesions. [ABSTRACT FROM AUTHOR]

Abstract: Induction of tenascin-C mRNA by cyclic strain in fibroblasts depends on RhoA and Rho dependent kinase (ROCK). Here we show that integrin-linked kinase (ILK) is required upstream of this pathway. In ILK-deficient fibroblasts, RhoA was not activated and tenascin-C mRNA remained low after cyclic strain; tenascin-C expression was unaffected by ROCK inhibition. In ILK wild-type but not ILK −/− fibroblasts, cyclic strain-induced reorganization of actin stress fibers and focal adhesions, as well as nuclear translocation of MAL, a transcriptional co-activator that links actin assembly to gene expression. These findings support a role for RhoA in ILK-mediated mechanotransduction. Rescue of ILK −/− fibroblasts by expression of wild-type ILK restored these responses to cyclic strain. Mechanosensation is not entirely abolished in ILK −/− fibroblasts, since cyclic strain activated Erk-1/2 and PKB/Akt, and induced c-fos mRNA in these cells. Conversely, lysophosphatidic acid stimulated RhoA and induced both c-fos and tenascin-C mRNA in ILK −/− cells. Thus, the signaling pathways controlling tenascin-C expression are functional in the absence of ILK, but are not triggered by cyclic strain. Our results indicate that ILK is selectively required for the induction of specific genes by mechanical stimulation via RhoA-mediated pathways. [Copyright &y& Elsevier]