Cells in an organism are subjected to numerous sources of external and internal forces, and are able to sense and respond to these forces. Integrin-mediated adhesion links the extracellular matrix outside cells to the cytoskeleton inside, and participates in sensing, transmitting and responding to forces. While integrin adhesion rapidly adapts to changes in forces in isolated migrating cells, it is not known whether similar or more complex responses occur within intact, developing tissues. Here, we studied changes in integrin adhesion composition upon different contractility conditions in Drosophila embryonic muscles. We discovered that all integrin adhesion components tested were still present at muscle attachment sites (MASs) when either cytoplasmic or muscle myosin II was genetically removed, suggesting a primary role of a developmental programme in the initial assembly of integrin adhesions. Contractility does, however, increase the levels of integrin adhesion components, suggesting a mechanism to balance the strength of muscle attachment to the force of muscle contraction. Perturbing contractility in distinct ways, by genetic removal of either cytoplasmic or muscle myosin II or eliminating muscle innervation, each caused unique alterations to the stoichiometry at MASs. This suggests that different integrin-associated proteins are added to counteract different kinds of force increase.

Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. Here, we examine how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produced relatively minor phenotypes, gain of vinculin activity, through a loss of head-tail autoinhibition, caused lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality was suppressed by competition with single vinculin-binding sites from talin. Activated Drosophila vinculin triggered the formation of cytoplasmic adhesion complexes through the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin were segregated into morphogenetic defects caused by its whole head domain and lethality caused by its D1 domain. These findings demonstrate the crucial importance of the tight control of the activity of vinculin.

Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. We show that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. We confirmed these alternatives by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, we show that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.