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Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced β3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the β3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of β3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the β3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked down for CFTR (CF transmembrane conductance regulator) were compared with control counterparts. We show that CFTR mutation in primary HAECs and CFTR knockdown cells promote the overexpression and oversecretion of TGF-β1 and DKK1 when cultured at an air–liquid interface. These dynamic changes result in hyperactivation of the TGF-β pathway and inhibition of the Wnt pathway through degradation of β-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-β and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-β, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF. [ABSTRACT FROM AUTHOR]

Integrin-dependent adhesion sites consist of clustered integrins that transmit mechanical forces and provide signaling required for cell survival and morphogenesis. Despite their importance, the regulation of integrin clustering by the cytoplasmic adapter protein talin (Tai) and phosphatidylinositol (PI)-4,5-biphosphate (PI(4,5)[P.sub.2]) lipids nor their dynamic coupling to the actin cytoskeleton is fully understood. By using a Tal-dependent integrin clustering assay in intact cells, we identified a Pl(4,5)[P.sub.2]-binding basic ridge spanning across the F2 and F3 domains of the Tai head that regulates integrin clustering. Clustering requires a new Pl(4,5)[P.sub.2]-binding site in F2 and is negatively regulated by autoinhibitory interactions between F3 and the Tai rod (Tai-R). The release of the Tal-R exposes a new [beta]3-integrin-binding site in F3, enabling interaction with a membrane proximal acidic motif, which involves the formation of salt bridges between [K.sup.316] and [K.sup.324] with [E.sup.726] and [D.sup.723], respectively. This interaction shields the [beta]-integrin tail from reassociation with its [alpha] subunit, thereby maintaining the integrin in a substrate-binding and clustering-competent form. doi/10.1083/jcb.200908134

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated [alpha]v[beta] integrins into focal adhesions. Manganese ([Mn.sup.2+]) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)[P.sub.2]). Fluorescence recovery after photo-bleaching analysis of [Mn.sup.2+]-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)[P.sub.2]. The dynamic remodeling of this ternary complex controls cell motility.

Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed [alpha] V [beta] 3-integrin dynamics in migrating cells using a green fluorescent protein-tagged [beta] 3-integrin chain. At the cell front, adhesion sites containing [alpha] V [beta] 3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of [beta] 3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast [beta] 3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.

Steel factor (SLF) and its cognate receptor, c-kit, have been implicated in the generation of melanocytes from migrating neural crest (NC) cells during early vertebrate embryogenesis. However, the source of SLF in the early avian embryo and its precise role in melanogenesis are unclear. We report here that NC cells themselves express and release SLF protein, which in turn acts as an autocrine factor to induce melanogenesis in nearby NC cells. These results indicate that NC cell subpopulations play an active role in the determination of their cell fate and suggest a different developmental role for the embryonic microenvironment than what has been previously proposed.