The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion

Cell adhesion requires linkage of transmembrane receptors to the cytoskeleton through intermediary linker proteins. Integrin-based adhesion to the extracellular matrix (ECM) involves large adhesion complexes that contain multiple cytoskeletal adapters that connect to the actin cytoskeleton. Many of these adapters, including the essential cytoskeletal linker Talin, have been shown to contain multiple actin-binding sites (ABSs) within a single protein. To investigate the possible role of having such a variety of ways of linking integrins to the cytoskeleton, we generated mutations in multiple actin binding sites in Drosophila talin. Using this approach, we have been able to show that different actin-binding sites in talin have both unique and complementary roles in integrin-mediated adhesion. Specifically, mutations in either the C-terminal ABS3 or the centrally located ABS2 result in lethality showing that they have unique and non-redundant function in some contexts. On the other hand, flies simultaneously expressing both the ABS2 and ABS3 mutants exhibit a milder phenotype than either mutant by itself, suggesting overlap in function in other contexts. Detailed phenotypic analysis of ABS mutants elucidated the unique roles of the talin ABSs during embryonic development as well as provided support for the hypothesis that talin acts as a dimer in in vivo contexts. Overall, our work highlights how the ability of adhesion complexes to link to the cytoskeleton in multiple ways provides redundancy, and consequently robustness, but also allows a capacity for functional specialization. The construction of tissues in animals requires cells to form attachments (adhesions), to other cells or the surrounding mixture of proteins and molecules known as the Extracellular matrix (ECM). Cell adhesion to the ECM has an important role in providing the mechanical stability that allows cells to arrange into different shapes during animal development. Key to this stability is their ability to connect to and organise the intracellular cytoskeleton such that it forms a scaffold that is both strong and flexible. To study the strategies used by cells to link the ECM to the cytoskeleton we introduced mutations into a protein called talin that is a key intermediary in adhesions between the outside environment and the cytoskeleton. Talin has different ways of linking to the actin cytoskeleton as it contains multiple domains that bind actin. Using a genetic approach, we disrupted these domains separately or together to block actin binding. We found these domains have both unique and complementary roles in cell-ECM adhesion indicating that having multiple actin-binding sites provides functional redundancy and specialization. Overall, our findings provide mechanistic insight into how cells can regulate cell adhesions by changing how they link to the cytoskeleton.