RhoA mediates stress fiber strain site recognition by the LIM protein testin

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize strained actin while the N-terminal domains alone recognize unstrained actin. Phosphorylation and cancer related mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes WT testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin9s mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state.