Investigating Expanded localization and binding partners. Leonie Alexandra Enderle, Robyn Rosenfeld, Vladimir Belozerov, Helen McNeill. Research, SLRI, Toronto, Ontario, Canada.

   The Hippo kinase pathway plays an important role in growth regulation during Drosophila development and is highly conserved between species. It is well known how the core pathway, consisting of the kinases Hippo and Warts and the adaptor proteins Salvador and Mats, negatively regulates the transcriptional coactivator Yorkie. Upstream regulation of the Hippo pathway, however, is less well understood. One upstream regulator is the FERM-domain protein Expanded which interacts with different components of the Hippo pathway at several levels. Expanded localizes to apical junctions where it can bind Yorkie to prevent it from entering the nucleus. Further, Expanded protein levels seem dependent on the correct localization of Expanded in the epithelium. We therefore seek to understand the mechanisms that regulate the apical and junctional localization of Expanded and their biological function. We are using the Drosophila larval eye imaginal disc to study Expanded localization in epithelia. We have investigated the behavior of different Expanded protein truncations and identified two distinct regions in the Expanded C-terminus that are required for junctional localization. Apical and junctional localization is regulated separately since truncations missing one or both of the identified domains were still enriched apically. Finally, we are performing Affinity Purification coupled to Mass Spectrometry with Expanded from cell lines and Drosophila embryonic tissue to identify novel binding partners.