Shear stress is a fundamental determinant of vascular homeostasis, regulating vascular remodelling, cardiac development and atherogenesis, but the mechanisms of transduction are poorly understood. Previous work showed that the conversion of integrins to a high-affinity state mediates a subset of shear responses, including cell alignment and gene expression. Here we investigate the pathway upstream of integrin activation. PECAM-1 (which directly transmits mechanical force), vascular endothelial cell cadherin (which functions as an adaptor) and VEGFR2 (which activates phosphatidylinositol-3-OH kinase) comprise a mechanosensory complex. Together, these receptors are sufficient to confer responsiveness to flow in heterologous cells. In support of the relevance of this pathway in vivo, PECAM-1-knockout mice do not activate NF-kappaB and downstream inflammatory genes in regions of disturbed flow. Therefore, this mechanosensing pathway is required for the earliest-known events in atherogenesis.

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.

Growth of normal cells is anchorage-dependent because signalling through multiple pathways including Erk, PI 3-kinase and Rac requires integrin-mediated cell adhesion 1 . Components of these pathways localize to low density, cholesterol-rich domains in the plasma membrane named "lipid rafts" 2 , 3 or "cholesterol enriched membrane microdomains" (CEMM) 4 . We previously reported that integrin-mediated adhesion regulates CEMM trafficking such that cell detachment from the extracellular matrix (ECM) triggers CEMM internalisation and clearance from the plasma membrane 5 . We now report that this internalisation is mediated by dynamin-2 and caveolin-1. Internalisation requires phosphorylation of caveolin-1 on tyrosine 14. A shift in localisation of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalisation upon cell detachment, which mediates inhibition of Erk, PI 3-kinase and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1. Keywordsanchorage-dependent cell growth; cancer; integrin signalling; caveolin; cholesterol-enriched membrane microdomains (CEMM); Rho GTPases Loss of anchorage dependence of growth in vitro is closely associated with tumour growth and metastasis in vivo 1 . The effects of integrins on multiple growth regulatory pathways mediate anchorage-dependence. Conversely, anchorage-independence in cancer cells is due to constitutive activation of these pathways such that integrin-mediated adhesion is no longer Supplementary Information accompanies the paper on the Nature Cell Biology's website. Competing Interests statementThe authors declare that they have no competing financial interests. The best defined subtype of CEMM is caveolae, which contain caveolin-1, or in muscle, caveolin-3 7 . Caveolae are ∼100 nm invaginations of the plasma membrane involved in clathrin-independent membrane traffic 8 and intracellular cholesterol transport 9 . Caveolin is a 21 kD protein first identified as a substrate for the v-src tyrosine kinase, which, among several other kinases, phosphorylates caveolin on tyr14 10 , 11 . Caveolin is also implicated in modulation of signal transduction. Caveolin inhibits a number of enzymes 11 , 12 and has been identified as a candidate tumour suppressor 13 -16 . Many tumour cells show loss of caveolin expression, and its re-expression reverses anchorage-independent growth 12 . 16% of human breast cancers contain a caveolin mutation 17 and caveolin knockout mice show dramatic acceleration of tumorigenesis in response to carcinogenic stimuli 15 , 16 . NIH Public AccessCaveolin is involved in internalisation of GM1 8 . We therefore investigated the role of caveolin in CEMM internalisation in anchorage-dependent cells after integrin signalling was inactivated by detaching cells from the ECM 1 . When cells were detached, caveolin-1 showed timedependent movement from the plasma membrane to an intracellular compartment ( Fig. 1a; results quantified in 1b) on the same time scale as the raft marker GM1 5 . This...

The alpha4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because alpha4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the alpha4 tail to identify molecules that could account for alpha4-specific signalling. Here we report that the alpha4 tail, but not several other alpha-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with alpha4 integrins in Jurkat T cells at high stoichiometry, and joining the alpha4 tail to alphaIIb resulted in a complex of integrin alphaIIbbeta3 with paxillin. This association markedly enhanced the rates of alphaIIbbeta3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the alpha4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, alpha4beta1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the alpha4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress-induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of¯ow. This directional Rac1 activation is downstream of shear-induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates ow-induced stimulation of nuclear factor kB (NF-kB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM-1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and in¯ammatory dysfunction of endothelial cells in atherosclerosis.