Alexander R. Dunn

E-cadherin is under constitutive actomyosin-generated tension that is increased at cell–cell contacts upon externally applied stretch

Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and αE-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell–cell contacts, although tension is further increased at cell–cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell–cell junctions but throughout the cell surface.

The minimal cadherin-catenin complex binds to actin filaments under force

INTRODUCTION Cadherins are an ancient class of transmembrane proteins that are essential for the formation of multicellular tissues in metazoans. Cadherins link intercellular adhesions to the cellular cytoskeleton, but how they are connected specifically to actin filaments is a hotly debated issue. Genetic and cell culture experiments indicate that E-cadherin, β-catenin, and the actin filament binding protein αE-catenin form a minimal cadherin-catenin complex that binds to the actin cytoskeleton directly in epithelial tissues. However, experiments with purified proteins showed that a stable cadherin-catenin complex can be reconstituted, but it does not bind strongly to actin filaments in solution. Nevertheless, cell culture experiments indicated that the cadherin-catenin complex is under constitutive actomyosin-generated tension and that this connection is required for mechanotransduction at cadherin-based adhesions. Here, we tested the hypothesis that tension is required to stabilize a linkage between the cadherin-catenin complex and actin filaments, and clarify how the cadherin-catenin complex could interact directly with the actin cytoskeleton in cells. Two-state catch bond model of cadherin-catenin/F-actin interactions. Force stabilizes the cadherin-catenin/F-actin bond by shifting it from a weakly to a strongly bound state. The force dependence of the connection between the cadherin-catenin complex and actin f laments may explain the mechanosensitivity of cadherin-mediated intercellular adhesions. RATIONALE We developed an optical trap–based assay to measure the lifetime of cadherin-catenin complex/actin filament bonds under tension. An actin filament was attached to two optically trapped beads and suspended above purified cadherin-catenin complexes immobilized on a glass coverslip surface that was precoated with glass microspheres. The coverslip was mounted on a motorized stage. This spatial arrangement was informed by electron tomography of cell-cell junctions, which showed actin filaments parallel to the plasma membrane. Tension was applied to cadherin-catenin complex/actin bonds by moving the sample stage back and forth parallel to the actin filament; if the immobilized cadherin-catenin complexes bound the actin filament, the attached beads were displaced from the optical trap. The lifetime of the bond was measured from the time series of the force exerted on the trapped beads. Kinetic models were fit to bond lifetime distributions with respect to applied force. RESULTS We observed robust, reproducible cadherin-catenin complex/actin filament binding under force in optical trap–based experiments. Bond lifetime distributions had a biphasic dependence on force. The mean lifetimes increased from ~60 ms at low force to ~1.2 s at ~10 pN, after which they decreased. A two-state catch bond model is consistent with the biphasic mean lifetime distribution and the presence of two distinct lifetime subpopulations. In this model, bonds between a cadherin-catenin complex and an actin filament form in a weakly bound state and quickly dissociate, but rapidly transition to a strongly bound state as applied force increases. Long lifetimes are achieved in this state until higher forces accelerate dissociation from the strongly bound state (see the figure). CONCLUSION Our data and kinetic model reconcile previous in vitro and in vivo work by demonstrating that the cadherin-catenin complex binds robustly to actin filaments under force. Thus, it seems that direct cadherin-catenin complex/actin filament binding was not detected in previous solution-based assays because bonds were not strengthened by tension. The two bound states in our model may correspond to different conformational states of αE-catenin, consistent with previous observations that αE-catenin may undergo changes in conformation in response to actomyosin-generated cytoskeletal tension. Our model of cadherin-catenin complex/ actin filament bond dissociation, combined with previous evidence of cooperative binding of αE-catenin to actin filaments, indicates that the linkage is self-reinforcing and that its stability is dynamically regulated by mechanical force during tissue development and maintenance. Pulling me apart only makes me stronger Tension transmitted between neighboring cells can exert profound effects on cell proliferation, differentiation, and tissue organization. Exactly how intercellular mechanical tension is sensed at the molecular level is unknown. One attractive hypothesis is that a linkage between the cell-cell adhesion molecule E-cadherin, its binding partners α- and β-catenin, and actin filaments may act as a tension sensor. However, how this linkage is established at the molecular level is not known. Buckley et al. used optical tweezers to determine how mechanical load influences interactions of the cadherin/catenin complex with single actin filaments. The data support a model in which force shifts the interaction from a force-independent, weakly bound state to a highly force-sensitive, strongly bound state. The findings may explain how cells maintain tissue integrity while still being able to move and change shape. Science, this issue p. 10.1126/science.1254211 A protein complex involved in cell adhesion forms a two-state catch bond with the cytoskeleton under mechanical load. Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)–actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap–based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

Influence of Perfluoroarene–Arene Interactions on the Phase Behavior of Liquid Crystalline and Polymeric Materials

A stabilization of the liquid-crystalline mesophase and thus an enlarged temperature range of the mesogenic phase is achieved by adding perfluorotriphenylene to a chiral liquid-crystalline triphenylene. This mesophase is based on 1:1 perfluoroarene-arene interactions (see picture). In a polymer with triphenylenes as mesogens in the side chains, the addition of perfluorotriphenylene led to crystallization.

Molecular Tension Sensors Report Forces Generated by Single Integrin Molecules in Living Cells

Living cells are exquisitely responsive to mechanical cues, yet how cells produce and detect mechanical force remains poorly understood due to a lack of methods that visualize cell-generated forces at the molecular scale. Here we describe Förster resonance energy transfer (FRET)-based molecular tension sensors that allow us to directly visualize cell-generated forces with single-molecule sensitivity. We apply these sensors to determine the distribution of forces generated by individual integrins, a class of cell adhesion molecules with prominent roles throughout cell and developmental biology. We observe strikingly complex distributions of tensions within individual focal adhesions. FRET values measured for single probe molecules suggest that relatively modest tensions at the molecular level are sufficient to drive robust cellular adhesion.

Mechanical Load Induces a 100-Fold Increase in the Rate of Collagen Proteolysis by MMP-1

Although mechanical stress is known to profoundly influence the composition and structure of the extracellular matrix (ECM), the mechanisms by which this regulation occurs remain poorly understood. We used a single-molecule magnetic tweezers assay to study the effect of force on collagen proteolysis by matrix metalloproteinase-1 (MMP-1). Here we show that the application of ∼10 pN in extensional force causes an ∼100-fold increase in proteolysis rates. Our results support a mechanistic model in which the collagen triple helix unwinds prior to proteolysis. The data and resulting model predict that biologically relevant forces may increase localized ECM proteolysis, suggesting a possible role for mechanical force in the regulation of ECM remodeling.

Velocity, processivity, and individual steps of single myosin V molecules in live cells.

We report the tracking of single myosin V molecules in their natural environment, the cell. Myosin V molecules, labeled with quantum dots, are introduced into the cytoplasm of living HeLa cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. We perform an intracellular measurement of key parameters of this molecular transporter: velocity, processivity, step size, and dwell time. Our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells.

Mechanical control of the sense of touch by β-spectrin

The ability to sense and respond to mechanical stimuli emanates from sensory neurons and is shared by most, if not all, animals. Exactly how such neurons receive and distribute mechanical signals during touch sensation remains mysterious. Here, we show that sensation of mechanical forces depends on a continuous, pre-stressed spectrin cytoskeleton inside neurons. Mutations in the tetramerization domain of Caenorhabditis elegans β-spectrin (UNC-70), an actin-membrane crosslinker, cause defects in sensory neuron morphology under compressive stress in moving animals. Through atomic force spectroscopy experiments on isolated neurons, in vivo laser axotomy and fluorescence resonance energy transfer imaging to measure force across single cells and molecules, we show that spectrin is held under constitutive tension in living animals, which contributes to elevated pre-stress in touch receptor neurons. Genetic manipulations that decrease such spectrin-dependent tension also selectively impair touch sensation, suggesting that such pre-tension is essential for efficient responses to external mechanical stimuli.

Probing the open state of cytochrome P450cam with ruthenium-linker substrates.

Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-Å deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.

Quantification of nanowire penetration into living cells

High-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.

Visualizing the Interior Architecture of Focal Adhesions with High-Resolution Traction Maps

Focal adhesions (FAs) are micron-sized protein assemblies that coordinate cell adhesion, migration, and mechanotransduction. How the many proteins within FAs are organized into force sensing and transmitting structures is poorly understood. We combined fluorescent molecular tension sensors with super-resolution light microscopy to visualize traction forces within FAs with <100 nm spatial resolution. We find that αvβ3 integrin selectively localizes to high force regions. Paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles as force transducers. These observations suggest that αvβ3 integrin and paxillin may play important roles in mechanotransduction.