In vivo regulation of integrin turnover by outside-in activation.

The development of three-dimensional tissue architecture requires precise control over the attachment of cells to the extracellular matrix (ECM). Integrins, the main ECM-binding receptors in animals, are regulated in multiple ways to modulate cell-ECM adhesion. One example is the conformational activation of integrins by extracellular signals ('outside-in activation') or by intracellular signals ('inside-out activation'), whereas another is the modulation of integrin turnover. We demonstrate that outside-in activation regulates integrin turnover to stabilize tissue architecture in vivo Treating Drosophila embryos with Mg(2+) and Mn(2+), known to induce outside-in activation, resulted in decreased integrin turnover. Mathematical modeling combined with mutational analysis provides mechanistic insight into the stabilization of integrins at the membrane. We show that as tissues mature, outside-in activation is crucial for regulating the stabilization of integrin-mediated adhesions. This data identifies a new in vivo role for outside-in activation and sheds light on the key transition between tissue morphogenesis and maintenance.

The talin head domain reinforces integrin-mediated adhesion by promoting adhesion complex stability and clustering.

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.

In vivo quantitative analysis of Talin turnover in response to force.

Cell adhesion to the extracellular matrix (ECM) allows cells to form and maintain three-dimensional tissue architecture. Cell-ECM adhesions are stabilized upon exposure to mechanical force. In this study, we used quantitative imaging and mathematical modeling to gain mechanistic insight into how integrin-based adhesions respond to increased and decreased mechanical forces. A critical means of regulating integrin-based adhesion is provided by modulating the turnover of integrin and its adhesion complex (integrin adhesion complex [IAC]). The turnover of the IAC component Talin, a known mechanosensor, was analyzed using fluorescence recovery after photobleaching. Experiments were carried out in live, intact flies in genetic backgrounds that increased or decreased the force applied on sites of adhesion. This analysis showed that when force is elevated, the rate of assembly of new adhesions increases such that cell-ECM adhesion is stabilized. Moreover, under conditions of decreased force, the overall rate of turnover, but not the proportion of adhesion complex components undergoing turnover, increases. Using point mutations, we identify the key functional domains of Talin that mediate its response to force. Finally, by fitting a mathematical model to the data, we uncover the mechanisms that mediate the stabilization of ECM-based adhesion during development.