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 pico-Newton (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.

SummaryDirectional 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.

Contact Inhibition of Locomotion was discovered by Abercrombie more than 50 years ago to describe the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction upon contact1,2. Its failure was suggested to contribute to malignant invasion3-6. 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 invasion6-8, exhibit 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, like 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 localise 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, present an explanation for coherent directional migration of group of cells and establish a novel role for non-canonical Wnt signalling.

Here, we report a direct interaction between the beta1 integrin cytoplasmic tail and Rab25, a GTPase that has been linked to tumor aggressiveness and metastasis. Rab25 promotes a mode of migration on 3D matrices that is characterized by the extension of long pseudopodia, and the association of the GTPase with alpha5beta1 promotes localization of vesicles that deliver integrin to the plasma membrane at pseudopodial tips as well as the retention of a pool of cycling alpha5beta1 at the cell front. Furthermore, Rab25-driven tumor-cell invasion into a 3D extracellular matrix environment is strongly dependent on ligation of fibronectin by alpha5beta1 integrin and the capacity of Rab25 to interact with beta1 integrin. These data indicate that Rab25 contributes to tumor progression by directing the localization of integrin-recycling vesicles and thereby enhancing the ability of tumor cells to invade the extracellular matrix.

Directed cell migration is crucial for development, but most of our current knowledge is derived from in vitro studies. We analyzed how neural crest (NC) cells migrate in the direction of their target during embryonic development. We show that the proteoglycan Syndecan-4 (Syn4) is expressed in the migrating neural crest of Xenopus and zebrafish embryos. Loss-of-function studies using an antisense morpholino against syn4 show that this molecule is required for NC migration, but not for NC induction. Inhibition of Syn4 does not affect the velocity of cell migration, but significantly reduces the directional migration of NC cells. Furthermore, we show that Syn4 and PCP signaling control the directional migration of NC cells by regulating the direction in which the cell protrusions are generated during migration. Finally, we perform FRET analysis of Cdc42, Rac and RhoA in vitro and in vivo after interfering with Syn4 and PCP signaling. This is the first time that FRET analysis of small GTPases has been performed in vivo. Our results show that Syn4 inhibits Rac activity, whereas PCP signaling promotes RhoA activity. In addition, we show that RhoA inhibits Rac in NC cells. We present a model in which Syn4 and PCP control directional NC migration by, at least in part, regulating membrane protrusions through the regulation of small GTPase activities.

SummaryCollective 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.

SummaryContact 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.

SummaryLittle is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.