Kallem, T.; Guo, Y.; Reynolds, M. K.; Ball, N. J.; Athale, M.; Baker, K. B.; Mykuliak, V. V.; Ek, F.; Aldaz-Casanova, S.; Turkki, P.; Hytonen, V. P.; Brown, N. H.; Hoffman, B. D.; Yan, J.; Goult, B. T.

Cells transmit force between the extracellular matrix and the actin cytoskeleton through integrin adhesion complexes centred on talin and vinculin. Vinculin binds talin through -helical vinculin-binding sites (VBS) that are exposed when talin rod domains unfold under force. Dissecting the significance of this interaction has relied heavily on the A50I mutation in vinculin, which has been widely used as a talin-binding-null mutant. Here we show that although the A50I mutation abolishes binding to the -catenin VBS, it retains nanomolar-affinity binding to multiple talin VBS. We therefore designed an improved mutant, I12K/A50I, that eliminates this residual talin binding. Biochemical assays and single-molecule stretching experiments demonstrate that I12K/A50I VD1 fails to bind talin even when VBS are exposed by force. Using vinculin tension and conformation sensors, we show that talin binding is required for efficient recruitment of vinculin to focal adhesions and for establishing spatial gradients of vinculin tension. However, vinculin can still experience mechanical load in the absence of talin binding. These results demonstrate that A50I is not a talin-binding-null and reveal that, while talin is not required for vinculin loading, it is essential for organising the spatial distribution of mechanical load within adhesion complexes.