Hippo Activation through Homo-dimerization and Membrane Association for Growth Inhibition and Organ Size Control. Yaoting Deng¹, Yurika Matsui², Yifan Zhang³, Zhi-Chun Lai^1,2,3,4. 1) Biochemistry and Molecular Biology, Penn State University, University Park, PA; 2) Intercollege Graduate Degree Program in Cell and Developmental Biology; 3) Intercollege Graduate Degree Program in Genetics; 4) Department of Biology.

   Hippo (Hpo) signaling plays a critical role in restricting tissue growth and organ size in both invertebrate and vertebrate animals. However, how the Hpo kinase is regulated during development has not been clearly understood. Using a Bimolecular Fluorescence Complementation (BiFC) assay, we have investigated the functional significance of Hpo homo-dimer formation and subcellular localization in living cells. We found that Hpo dimerization and membrane association are both critical for its activation in growth inhibition. As dimerization facilitates Hpo to access its binding partner, Hpo kinases in the homo-dimer trans-phosphorylate each other to increase their enzymatic activity. Moreover, loss- and gain-of-function studies indicate that upstream regulators, Expanded, Merlin and Kibra, play a critical role in promoting Hpo dimerization as well as association to the cell membrane. Enforced Hpo localization to the cell membrane increases Hpo dimerization and its activity. Therefore, homo-dimerization and membrane localization are two important mechanisms for Hpo activation in growth control during animal development.