FUNCTIONAL CHARACTERIZATION OF FORMIN-DEPENDENT ACTIN POLYMERIZATION AT ADHERENS JUNCTIONS

Ph.DPH.D. IN MECHANOBIOLOGY (FOS

An optogenetic tool for the activation of endogenous diaphanous-related formins induces thickening of stress fibers without an increase in contractility: Photo-activation of Diaphanous-related Formins

We have developed an optogenetic technique for the activation of diaphanous related formins. Our approach is based on fusion of the Light-Oxygen-Voltage 2 domain of Avena sativa Phototrophin1 to an isolated Diaphanous Autoregulatory Domain from mDia1. This “caged” diaphanous autoregulatory domain was inactive in the dark, but in the presence of blue light rapidly activated endogenous diaphanous related formins. Using an F-actin reporter we observed filopodia and lamellipodia formation as well as a steady increase in F-actin along existing stress fibers, starting within minutes of photo-activation. Interestingly, we did not observe the formation of new stress fibers. Remarkably, a 1.9 fold increase in F-actin was not paralleled by an increase in myosin II along stress fibers and the amount of tension generated by the fibers, as judged by focal adhesion size, appeared unchanged. Our results suggest a decoupling between F-actin accumulation and contractility in stress fibers and demonstrate the utility of photoactivatable diaphanous autoregulatory domain for the study of diaphanous related formin function in cells

E-cadherin interactome complexity and robustness resolved by quantitative proteomics

E-cadherin–mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness mediated by E-cadherin is conferred by its extracellular cadherin domains and is regulated by an assembly of intracellular adaptors and enzymes associated with its cytoplasmic tail. We used proximity biotinylation and quantitative proteomics to identify 561 proteins in the vicinity of the cytoplasmic tail of E-cadherin. In addition, we used proteomics to identify proteins associated with E-cadherin–containing adhesion plaques from a cell-glass interface, which enabled the assignment of cellular localization to putative E-cadherin–interacting proteins. Moreover, by tagging identified proteins with GFP (green fluorescent protein), we determined the subcellular localization of 83 putative E-cadherin–proximal proteins and identified 24 proteins that were previously uncharacterized as part of adherens junctions. We constructed and characterized a comprehensive E-cadherin interaction network of 79 published and 394 previously uncharacterized proteins using a structure-informed database of protein-protein interactions. Finally, we found that calcium chelation, which disrupts the interaction of the extracellular E-cadherin domains, did not disrupt most intracellular protein interactions with E-cadherin, suggesting that the E-cadherin intracellular interactome is predominantly independent of cell-cell adhesion