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Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P 2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of v integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor -1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model (mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage. [ABSTRACT FROM AUTHOR]

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells ( Sharpin cpdm), and mice with a stromal ( S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin cpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin cpdm mice displayed reduced collagen arrangement and extracellular matrix ( ECM) stiffness. Moreover, Sharpin cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM. [ABSTRACT FROM AUTHOR]

Fibrillar adhesions are important structural and adhesive components in fibroblasts, and are required for fibronectin fibrillogenesis. While nascent and focal adhesions are known to respond to mechanical cues, the mechanoresponsive nature of fibrillar adhesions remains unclear. Here, we used ratiometric analysis of paired adhesion components to determine an appropriate fibrillar adhesion marker. We found that active α5β1-integrin exhibits the most definitive fibrillar adhesion localization compared to other proteins, such as tensin-1, reported to be in fibrillar adhesions. To elucidate the mechanoresponsiveness of fibrillar adhesions, we designed a cost-effective and reproducible technique to fabricate physiologically relevant stiffness gradients on thin polyacrylamide (PA) hydrogels, embedded with fluorescently labelled beads. We generated a correlation curve between bead density and hydrogel stiffness, thus enabling a readout of stiffness without the need for specialized knowhow, such as atomic force microscopy (AFM). We find that stiffness promotes growth of fibrillar adhesions in a tensin-1-dependent manner. Thus, the formation of these extracellular matrix-depositing structures is coupled to the mechanical parameters of the cell environment and may enable cells to fine-tune their matrix environment in response to changing physical conditions. [ABSTRACT FROM AUTHOR]

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes. [ABSTRACT FROM AUTHOR]