Summary The cytosolic protein α–catenin is a postulated force-transducer at cadherin complexes [1]. The demonstration of force activation, identification of consequent downstream events in live cells, and development of tools to study these dynamic processes in living cells are central to elucidating the role of α–catenin in cellular mechanics and tissue function [2–10]. Here we demonstrate that α–catenin is a force-activatable mechano-transducer at cell-cell junctions, using an engineered α-catenin conformation sensor, based on fluorescence resonance energy transfer (FRET). This sensor reconstitutes α-catenin-dependent functions in α-catenin depleted cells, and recapitulates the behavior of the endogenous protein. Dynamic imaging of cells expressing the sensor demonstrated that α-catenin undergoes immediate, reversible conformational switching, in direct response to different mechanical perturbations of cadherin adhesions. Combined magnetic twisting cytometry with dynamic FRET imaging [11] revealed rapid, local conformational switching, upon the mechanical stimulation of specific cadherin bonds. At acutely stretched cell-cell junctions, the immediate, reversible conformational change further reveals that α-catenin behaves like an elastic spring in series with cadherin and actin. The force-dependent recruitment of vinculin—a principal α-catenin effector—to junctions requires the vinculin-binding-site of the α–catenin sensor [1, 12–16]. In cells, the relative rates of force-dependent α–catenin conformation switching and vinculin recruitment reveal that α–catenin activation and vinculin recruitment occur sequentially rather than in a concerted process, with vinculin accumulation being significantly slower. This engineered α-catenin sensor revealed that α–catenin is a reversible, stretch-activatable sensor that mechanically links cadherin complexes and actin, and is an indispensable player in cadherin-specific mechano-transduction at intercellular junctions.

This report elucidates an E-cadherin-based force-transduction pathway that triggers changes in cell mechanics through a mechanism requiring epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), and the downstream formation of new integrin adhesions. This mechanism operates in addition to local cytoskeletal remodeling triggered by conformational changes in the E-cadherin-associated protein α-catenin, at sites of mechanical perturbation. Studies using magnetic twisting cytometry (MTC), together with traction force microscopy (TFM) and confocal imaging identified force-activated E-cadherin-specific signals that integrate cadherin force transduction, integrin activation and cell contractility. EGFR is required for the downstream activation of PI3K and myosin-II-dependent cell stiffening. Our findings also demonstrated that α-catenin-dependent cytoskeletal remodeling at perturbed E-cadherin adhesions does not require cell stiffening. These results broaden the repertoire of E-cadherin-based force transduction mechanisms, and define the force-sensitive signaling network underlying the mechano-chemical integration of spatially segregated adhesion receptors.

The findings presented here demonstrate the role of a-catenin in cadherin-based adhesion and mechanotransduction in different mechanical contexts. Bead-twisting measurements in conjunction with imaging, and the use of different cell lines and a-catenin mutants reveal that the acute local mechanical manipulation of cadherin bonds triggers vinculin and actin recruitment to cadherin adhesions in an actin-and a-catenin-dependent manner. The modest effect of a-catenin on the two-dimensional binding affinities of cell surface cadherins further suggests that forceactivated adhesion strengthening is due to enhanced cadherincytoskeletal interactions rather than to a-catenin-dependent affinity modulation. Complementary investigations of cadherin-based rigidity sensing also suggest that, although a-catenin alters traction force generation, it is not the sole regulator of cell contractility on compliant cadherin-coated substrata.

When microtubules are severed by laser ablation, newly created minus ends increase in curvature, but they straighten when dynein is inhibited. It is found that cytoplasmic dynein generates tension and friction along microtubule lengths and that these forces govern the dynamics of centrosome centering.

In living cells, a fluctuating torque is exerted on the nuclear surface but the origin of the torque is unclear. In this study, we found that the nuclear rotation angle is directionally persistent on a time scale of tens of minutes, but rotationally diffusive on longer time scales. Rotation required the activity of the microtubule motor dynein. We formulated a model based on microtubules undergoing dynamic instability, with tensional forces between a stationary centrosome and the nuclear surface mediated by dynein. Model simulations suggest that the persistence in rotation angle is due to the transient asymmetric configuration of microtubules exerting a net torque in one direction until the configuration is again randomized by dynamic instability. The model predicts that the rotational magnitude must depend on the distance between the nucleus and the centrosome. To test this prediction, rotation was quantified in patterned cells in which the cell's centrosome was close to the projected nuclear centroid. Consistent with the prediction, the angular displacement was found to decrease in these cells relative to unpatterned cells. This work provides the first mechanistic explanation for how nuclear dynein interactions with discrete microtubules emanating from a stationary centrosome cause rotational torque on the nucleus.

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus.

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This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper.