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 properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered; in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB). Membrane order at focal adhesion depends partly on phosphorylation of caveolin1 at Tyr14, which localizes to focal adhesions. Detachment of cells from the substratum triggers a rapid, caveolin-independent decrease in membrane order, followed by a slower, caveolin-dependent decrease that correlates with internalization of CtxB-stained domains. Endocytosed CtxB domains also become more fluid. Thus, membrane order is highly dependent on caveolae and focal adhesions. These results show that lipid raft properties are conferred by assembly of specific protein complexes. The ordered state within focal adhesions may have important consequences for signaling at these sites.

SUMMARYIntegrin-mediated adhesion regulates Rac1 membrane binding sites within lipid rafts. Detachment of cells from the substratum triggers clearance of rafts from the plasma membrane through caveolindependent internalization. The small GTPase Arf6 and microtubules also regulate Rac-dependent cell spreading and migration but the mechanisms are poorly understood. We now show that endocytosis of rafts after detachment requires F-actin, followed by microtubule-dependent trafficking to recycling endosomes (RE). When cells are replated on fibronectin, rafts exit from RE in an Arf6-dependent manner and return to the plasma membrane along microtubules. Both of these steps are required for plasma membrane targeting and activation of Rac1. These data therefore define a novel membrane raft trafficking pathway that is crucial for anchorage-dependent signaling.

Dextran vesicular nanoscaffolds were developed based on polysaccharide and renewable resource alkyl tail for dual encapsulation of hydrophilic and hydrophobic molecules (or drugs) and delivery into cells. The roles of the hydrophobic segments on the molecular self-organization of dextran backbone into vesicles or nanoparticles were investigated in detail. Dextran vesicles were found to be a unique dual carrier in which water-soluble molecules (like Rhodamine-B, Rh-B) and polyaromatic anticancer drug (camptothecin, CPT) were selectively encapsulated in the hydrophilic core and hydrophobic layer, respectively. The dextran vesicles were capable of protecting the plasma-sensitive CPT lactone pharmacophore against the hydrolysis by 10× better than the CPT alone in PBS. The aliphatic ester linkage connecting the hydrophobic tail with dextran was found to be cleaved by esterase under physiological conditions for fast releasing of CPT or Rh-B. Cytotoxicity of the dextran vesicle and its drug conjugate were tested on mouse embryonic fibroblast cells (MEFs) using MTT assay. The dextran vesicular scaffold was found to be nontoxic to living cells. CPT loaded vesicles were found to be 2.5-fold more effective in killing fibroblasts compared to that of CPT alone in PBS. Confocal microscopic images confirmed that both Rh-B and CPT loaded vesicles to be taken up by fibroblasts compared to CPT alone, showing a distinctly perinuclear localization in cells. The custom designed dextran vesicular provides new research opportunities for dual loading and delivering of hydrophilic and hydrophobic drug molecules.

Summary Anchorage-dependence of cell growth is a key metastasis-suppression mechanism that is mediated by effects of integrins on growth signaling pathways [1]. The small GTPase RalA is activated in metastatic cancers through multiple mechanisms and specifically induces anchorage independence [2–4]. Loss of integrin-mediated adhesion triggers caveolin-dependent internalization of cholesterol- and sphingolipid- rich lipid raft microdomains to the recycling endosomes; these domains serve as platforms for many signaling pathways and their clearance from the plasma membrane (PM) after cell detachment suppresses growth signaling [5, 6]. Conversely, re-adhesion triggers their return to the PM and restores growth signaling. Activation of Arf6 by integrins mediates exit of raft markers from the RE but is not sufficient for return to the PM. We now show that RalA but not RalB mediates integrin-dependent membrane raft exocytosis through the exocyst complex. Constitutively active RalA restores membrane raft targeting to promote anchorage independent growth signaling. Ras-transformed pancreatic cancer cells also show RalA-dependent constitutive PM raft targeting. These results identify RalA as a key determinant of integrin-dependent membrane raft trafficking and regulation of growth signaling. They therefore define a mechanism by which RalA regulates anchorage dependence and provide a new link between integrin signaling and cancer.