A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo

Cells in developing organisms are subjected to particular mechanical forces that shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Forster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically relevant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle-tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo. Author summary Cells in our body are constantly exposed to mechanical forces, which they need to sense and react to. In previous studies, fluorescent force sensors were developed to demonstrate that individual proteins in adhesion structures of a cell experience forces in the piconewton (pN) range. However, these cells were analyzed in isolation in an artificial plastic or glass environment. Here, we explored forces on adhesion proteins in their natural environment within a developing animal and used the muscle-tendon tissue in the fruit fly Drosophila as a model system. We made genetically modified fly lines with force sensors or controls inserted into the gene that produces the essential adhesion protein Talin. Using these force sensor flies, we found that only a small proportion of (<15%) of all the Talin proteins present at developing muscle-tendon attachments experience detectable forces at the same time. Nevertheless, a large amount of Talin is accumulated at these attachments during fly development. We found that this large Talin pool is important to prevent rupture of the muscle-tendon connection in adult flies that produce high muscle forces during flight. In conclusion, we demonstrated that a large pool of Talin proteins is required for stable muscle-tendon attachment, likely with the individual Talin molecules dynamically sharing the mechanical load.