The ability of cells to respond to physical forces is fundamental to development and physiology, including regulation of blood pressure, cell adhesion and migration. A major limitation to the study of these phenomena has been the difficulty of measuring molecular forces in cells in vivo. Grashoff et al. now report the development of a genetically encoded, fluorescent tension-sensing module capable of measuring mechanical forces across specific proteins in vivo. The sensor was tested on vinculin, a membrane-cytoskeletal protein that is recruited to focal adhesions and connects cell-adhesion molecules (integrins) to actin filaments. The data reveal a regulatory mechanism in which the ability of vinculin to bear force determines whether focal adhesions assemble or disassemble under force. This new biosensor should be applicable to other proteins involved in mechanotransduction. The ability of cells to respond to physical forces is central to development and physiology, but until now it has been difficult to directly measure forces across proteins in vivo. Here, however, a calibrated biosensor is described that can measure forces with high sensitivity across specific proteins in cells. This is applied to the vinculin protein, and a regulatory mechanism is revealed in which the force applied to vinculin determines whether focal adhesions assemble or disassemble. Mechanical forces are central to developmental, physiological and pathological processes1. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy2. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent3. We show that tension across vinculin in stable FAs is ∼2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

The phenomenon of contact inhibition of cell movement (or locomotion) was first identified more than 50 years ago in fibroblast cells in vitro, and defective contact inhibition is suggested as a factor in malignant cell invasiveness. It occurs when two cells touch; they retract their protrusions and change their direction of movement. But the molecular basis of this inhibition, and whether it happens in vivo, are still matters of controversy. Now time-lapse microscopy of neural crest cells, highly migratory cells of embryonic origin, has been used to demonstrate contact inhibition of locomotion both in vivo and in vitro, and that it can account for their directional migration. But when a neural crest cell meets another cell type it fails to display contact inhibition of locomotion, allowing it to invade the tissue. This paper studies a phenomenon called contact inhibition of locomotion, whereby fibroblast cells grown in cell culture retract their protrusions and change their direction on contact. It is shown that this occurs in vivo, and the molecular basis is revealed. Neural crest cells, highly migratory cells of embryonic origin, exhibit contact inhibition of locomotion both in vivo and in vitro, which accounts for their directional migration. However, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion, allowing it to invade the tissue. Contact inhibition of locomotion was discovered by Abercrombie more than 50 years ago and describes the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction on contact1,2. Its failure was suggested to contribute to malignant invasion3,4,5,6. However, the molecular basis of contact inhibition of locomotion and whether it also occurs in vivo are still unknown. Here we show that neural crest cells, a highly migratory and multipotent embryonic cell population, whose behaviour has been likened to malignant invasion6,7,8, demonstrate contact inhibition of locomotion both in vivo and in vitro, and that this accounts for their directional migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion; instead, it invades the other tissue, in the same manner as metastatic cancer cells3,5,9. We show that inhibition of non-canonical Wnt signalling abolishes both contact inhibition of locomotion and the directionality of neural crest migration. Wnt-signalling members localize at the site of cell contact, leading to activation of RhoA in this region. These results provide the first example of contact inhibition of locomotion in vivo, provide an explanation for coherent directional migration of groups of cells and establish a previously unknown role for non-canonical Wnt signalling.

Directional collective migration is now a widely recognized mode of migration during embryogenesis and cancer. However, how a cluster of cells responds to chemoattractants is not fully understood. Neural crest cells are among the most motile cells in the embryo, and their behavior has been likened to malignant invasion. Here, we show that neural crest cells are collectively attracted toward the chemokine Sdf1. While not involved in initially polarizing cells, Sdf1 directionally stabilizes cell protrusions promoted by cell contact. At this cell contact, N-cadherin inhibits protrusion and Rac1 activity and in turn promotes protrusions and activation of Rac1 at the free edge. These results show a role for N-cadherin during contact inhibition of locomotion, and they reveal a mechanism of chemoattraction likely to function during both embryogenesis and cancer metastasis, whereby attractants such as Sdf1 amplify and stabilize contact-dependent cell polarity, resulting in directional collective migration.

Collective cell migration is a mode of movement crucial for morphogenesis and cancer metastasis. However, little is known about how migratory cells coordinate collectively. Here we show that mutual cell-cell attraction (named here coattraction) is required to maintain cohesive clusters of migrating mesenchymal cells. Coattraction can counterbalance the natural tendency of cells to disperse via mechanisms such as contact inhibition and epithelial-to-mesenchymal transition. Neural crest cells are coattracted via the complement fragment C3a and its receptor C3aR, revealing an unexpected role of complement proteins in early vertebrate development. Loss of coattraction disrupts collective and coordinated movements of these cells. We propose that coattraction and contact inhibition act in concert to allow cell collectives to self-organize and respond efficiently to external signals, such as chemoattractants and repellents.

Contact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes to malignant invasion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investigate what determines this differential behavior, we study neural crest cells, a migratory stem cell population whose invasiveness has been likened to cancer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercellular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that contributes to CIL in cells in vivo.