The authors note that that in the constructs EcadTSMod, EcadTSModΔcyto, and EcadTSModΔmTFP, the Förster resonance energy transfer (FRET) acceptor fluorophore was monomeric enhanced yellow fluorescent protein (mEYFP)-not Venus as was originally reported. The photophysical properties of mEYFP and Venus, including absorption spectra, emission spectra, and fluorescence quantum yields are highly similar, leading to negligible changes in the Förster radius with the FRET donor monomeric Teal Fluorescent Protein (mTFP) (1). This substitution thus does not affect the FRET data reported in the original manuscript or their interpretation. The protein mEYFP is based on EYFP (Clontech) with the addition of the mutation A206K to suppress dimerization (2). www.pnas.org/cgi

Under ordinary circumstances, the membrane tension of a giant unilamellar vesicle is essentially nil. Using visible light, we stretch the vesicles, increasing the membrane tension until the membrane responds by the sudden opening of a large pore (several micrometers in size). Only a single pore is observed at a time in a given vesicle. However, a cascade of transient pores appear, up to 30-40 in succession, in the same vesicle. These pores are transient: they reseal within a few seconds as the inner liquid leaks out. The membrane tension, which is the driving force for pore opening, is relaxed with the opening of a pore and the leakage of the inner liquid; the line tension of the pore's edge is then able to drive the closure of a pore. We use fluorescent membrane probes and real-time videomicroscopy to study the dynamics of the pores. These can be visualized only if the vesicles are prepared in a viscous solution to slow down the leakout of the internal liquid. From measurements of the closure velocity of the pores, we are able to infer the line tension,. We have studied the effect of the shape of inclusion molecules on. Cholesterol, which can be modeled as an inverted cone-shaped molecule, increases the line tension when incorporated into the bilayers. Conversely, addition of cone-shaped detergents reduces. The effect of some detergents can be dramatic, reducing by two orders of magnitude, and increasing pore lifetimes up to several minutes. We give some examples of transport through transient pores and present a rough measurement of the leakout velocity of the inner liquid through a pore. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for biological systems and biotechnological applications.

During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell-ECM and cell-cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin-and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of αE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin-mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin-and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.D uring development, cohesive tissues exhibit extensive rearrangements that range from en masse migration, such as in wound healing (1), to complex local cell rearrangements, such as cell intercalation (2). In extreme cases in development (3) and in diseases such as metastatic cancers (4), tissue cohesion is lost and single-cell migration enabled, which results in cells populating distant sites. These morphogenetic processes reveal the importance of a fine coregulation, or crosstalk, between tissue cohesion (cadherin-based cell-cell adhesion) and cell migration [integrinbased extracellular matrix (ECM) adhesion] in the maintenance of tissue integrity and function.Interest in the crosstalk between cell-cell adhesion and cell migration dates back to the pioneering studies of Abercrombie and Heaysman in the 1950s (5, 6) and even earlier (7). Abercrombie coined the term "contact inhibition" to describe how cell-cell interactions between fibroblasts initially inhibited and then redirected their migration. Whether cell-cell contact inhibition of cell migration results from cell-cell contact-dependent spatial redistribution or down-regulation of the cell migration machinery, or both remains unknown.A major component of inte...

We discuss the pulling force f required to extrude a lipid tube from a living cell as a function of the extrusion velocity L . The main feature is membrane friction on the cytoskeleton. As recently observed for neutrophils, the tether force exhibits a ''shear thinning'' response over a large range of pulling velocities, which was previously interpreted by assuming viscoelastic flows of the sliding membrane. Here, we propose an alternative explanation based on purely Newtonian flow: The diameter of the tether decreases concomitantly with the increase of the membrane tension in the lipid tube. The pulling force is found to vary as L 1/3 , which is consistent with reported experimental data for various types of cells.cytoskeleton ͉ dynamics ͉ membrane tethers M any cellular processes [such as intracellular trafficking (1) or intercellular organelle transport (2)] involve the formation of thin tubular structures known as tethers. Membranous tails also are observed to be left by migrating cells in culture dishes (3). Tethers can be extracted from synthetic vesicles or living cells by the application of an external point force [using a fluid drag (4, 5) or pipette-tweezer system (6, 7)]. In the case of living cells, where the lipidic membrane is coupled to a cytoskeleton, tethers can be used as membrane sensors to measure the membrane-cytoskeleton adhesion energy W 0 (8-12).Our aim here is to describe the formation of a tether and to derive the required pulling force as a function of the extrusion velocity. Pulling a tube from a cell membrane implies a surface flow of lipid from the cell body to the tether through the membranecytoskeleton binders. This viscous drag gives rise to an increase of the membrane tension in the tube and a decrease of its radius. Statics of ExtrusionWe follow the thermodynamic analysis of tether formation proposed by Waugh and coworkers (7,13,14), Evans and Yeung (15), and Derényi and coworkers (16) for lipidic bilayers and extended to cell membranes by Sheetz and coworkers (10, 12). As shown in Fig. 1, the cell is usually held by micropipette suction (pressure Ϫ⌬P) that sets the membrane tension of the cell : 2 Ӎ R p ⌬P, where R p is the radius of the micropipette. The length of the tongue in the pipette is L p . Another case of experimental interest is found when cells are spread onto an adhesive surface, which ensures that the membrane tension remains weak or constant during the time course of the experiment. The tube is then either extruded by pulling out a small bead adhering to the membrane via micromanipulation if the cell is firmly adhered or, more simply, by applying a hydrodynamic flow over the cell in case of a discrete and sparse adhesion site. The length of the tube is L, the membrane tension of the tube is t , and its radius is r t . We want to relate r t and t to , the tension of the cell body, and to W 0 , the adhesion energy of the membrane to the cytoskeleton. The pulling force f 0 is deduced from the following four equations. The distribution of areas:2 r t dL ϭ 2 R p dL p...