Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.

Abstract. Functional differentiation in mammary epithelia requires specific hormones and local environmental signals . The latter are provided both by extracellular matrix and by communication with adjacent cells, their action being intricately connected in what appears to be a cascade of events leading to milk production . To distinguish between the influence of basement membrane and that of cell-cell contact in this process, we developed a novel suspension culture assay in which mammary epithelial cells were embedded inside physiological substrata . Single cells, separated from each other, were able to assimilate information from a laminin-rich basement membrane substratum and were induced to express 0-casein . In contrast, a stromal environment of collagen I was not sufficient to REGULATION of differentiation in complex tissues is determined not only by growth factors and hormones, but also by intercellular communication and by inter actions between cells and their extracellular matrix (Stoker et al., 1990) . In epithelial tissue, part ofthe extracellular matrix (ECM) I occurs in the form of a basement membrane, which provides positional information for cells and cues for organizing intracellular structure, as well as signals that regulate cellular behaviour. An ideal model for studying how ECM signals are transduced to control tissue-specific function is the simple epithelium of mammary gland . In adult animals, the cells undergo developmental changes during pregnancy and become highly secretory at lactation . The great advantage of this system is that in culture, mammary epithelial cells regain their differentiated phenotype only under suitable hormonal and substratum conditions . The model can therefore be used to understand the mechanism by which tissue-specific genes are expressed (Bissell and Hall, 1987;Streuli and Bissell, 1991) .Considerable evidence now indicates that basement membrane plays a significant role in regulating mammary phenotype. In vivo, the alveolar epithelium of rodent mammary 1 . Abbreviations used in this paper: DAPI, 4,6-diamidino-2-phenylindole ; ECM, extracellular matrix; EHS, Engelbreth-Holm-Swarm .® The Rockefeller University Press, 0021-9525/91/12/1383/13 $2 .00

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

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