Optogenetic tool for photoactivating diaphanous related formins [completed project]

Our interest in diaphanous related formins, such as mDia, stems from observations that mDia activity at adherens junctions has an impact on actin dynamics and cadherin concentration. However, there are conflicting reports in the literature. A major difficulty in assessing the function of mDia at cell-cell junctions is the lack of a reagent to activate endogenous mDia in the cell. Current approaches usually entail over expression of truncated constructs that are constitutively active, but do not necessarily localize correctly. We sought to develop a tool that would allow us to active endogenous mDia on demand. To this end we turned to optogenetics. In collaboration with Pei Hsuan and Klaus Hahn (UNC-Chapel Hill, NC) we have developed a photoactivatable Dia Autoinhibitory Domain (DAD), based on the fusion of a light oxygen voltage (LOV) domain to the DAD domain of mDia1. Our experiments have shown this to be a powerful tool for the activation of diaphanous related formins. For example, we discovered that when formins are activated in fibroblasts, actin polymerization takes place all along existing stress fibers and surprisingly the level of myosin in the stress fibers did not change. This work has been published in Cytoskeleton (Hoboken). 2013 Jul;70(7):394-407. doi: 10.1002/cm.21115. The plasmids for PA-DAD as well as full length constitutively active mutants are available from Addgene.