It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G 1͞S checkpoint and maintain high levels of p27 KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27 KIP1 is downregulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin ␣2␤1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.T he extracellular matrix (ECM) is a complex structure of collagens, glycoproteins, elastin, and proteoglycans that, in addition to providing a scaffold for tissues, regulates many fundamental cellular processes such as proliferation, survival, migration, and differentiation (1, 2). It is now appreciated that the ECM provides a complex combination of insoluble signals that, in concert with cell-cell contacts and soluble signals provided by growth factors, affect gene expression and influence cell functions (3-5). It is known that normal cells need contact with the ECM to progress into the cell cycle, and loss of this requirement is a hallmark of malignant cells (6). The mechanisms by which the ECM regulates the growth of anchoragedependent cells have received renewed attention over the past years and have been the subject of investigations that have pointed to cell cycle regulatory proteins that are influenced by contact between cells and the ECM (7-9). On binding to specific cell-surface receptors such as integrins, the ECM exerts its control on cell proliferation by modulating the expression of G 1 regulators in a growth factor-dependent and independent manner. In most conditions, there is cooperative action between growth factors and ECM that results in nonredundant signals that transcriptionally up-regulate cyclins D and E and downregulate the cyclin-dependent kinase (CDK) inhibitors p21 CIP1 , p27 KIP1 , and p57 KIP2 (10). This cooperative action permits cells to pass through the G 1 restriction point and to complete the cell cycle. However, there are other conditions where the ECM has been shown to send growth inhibitory rather than growth stimulatory signals. These conditions depend not only on the composition of the ECM but also on its physical state. Molecules of type I collagen in vivo are organized into striated fibrils. These structures, which are responsible for the resistance of many tissues, are lost on proteolytic degradation of the collagen. ...

The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.

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Supplementary key words toxin • His-mCherry-NT-lysenin • lateral membrane heterogeneity • vital confocal imaging • membrane tension • cholesterol • temperatureLipids at the outer leafl et of the mammalian plasma membrane are mainly composed of: i ) SM, the most abundant sphingolipid (SL), based on a ceramide backbone and bearing a phosphocholine polar head; ii ) glycosphingolipids (GSLs), another group of SLs bearing various sugars instead of phosphocholine, from simple glucosylceramide (GlcCer) to complex GSLs such as GM1 [for a review, see ( 1 )]; iii ) phosphatidylcholine (PC), the major glycerophospholipid, sharing the same phosphocholine polar head as SM; and iv ) nonpolar cholesterol. Lipid bilayers are no longer considered as a homogenous solvent for membrane proteins ( 2 ), but are now represented with lateral heterogeneity at two different scales of time and space: i ) transient nanometric "lipid rafts", defi ned as small clusters enriched in SLs, sterol, and GPI-anchored proteins ( 3, 4 ); versus ii ) submicrometric/mesoscale domains ( 5-15 ). These larger and more stable domains are well-characterized on artifi cial vesicles ( 16, 17 ), but their relevance for living cells has been questioned ( 18,19 ).The occurrence in living cells of submicrometric/mesoscale domains was fi rst inferred from unexpected behavior in fl uorescence recovery after photobleaching (FRAP)