SGK sensors for in vitro / in vivo dissection of signaling controlling membrane dynamics
Prof. Dr. Ralf Baumeister (Institute of Biology III - Bioinformatics and Molecular Genetics, University of Freiburg)
Project Summary Alterations in membrane trafficking between ER, Golgi, lysosome, but also mitochondria and the plasma membrane are beginning to emerge as causative for Alzheimer’s and Parkinson’s disease and modify signaling involved in tumor biology. In the previous funding period of BIOSS, we developed sensors and readouts in both C. elegans and tumor cell cultures to monitor altered membrane trafficking within the cell and investigated its consequences for intra- and intercellular signaling. We identified the AGC family kinase SGK-1 (serum-and-glucocorticoid inducible kinase 1) as a key regulator of membrane trafficking and protein complex assembly at the membrane. SGK-1 localization itself is dynamic and integrates activation by both insulin/IGF and mTORC pathways. Moreover, SGK-1 modulates FOXO shuttling in and out of the nucleus, which, in a negative feedback loop, controls SGK-1 expression. This feedback loop responds to nutrients and steroid/hormones, but also to osmoregulatory inputs controlling not only fat metabolism, stress responses, and aging, but also modulates tumor and neurodegeneration through affecting intracellular membrane trafficking in a yet non-understood way.
Now, we want to establish synthetic optical SGK-1 sensors to monitor: A) SGK-1 dynamic translocation between organelles and membranes and associated protein complexes. B) We will combine these read-outs, using our newly developed CRISPR/Cas9 technology, with SGK-1 variants in which either insulin/IGF or mTORC2 signaling has been eliminated, to resolve the contribution of each signaling component. We will apply these tools for worms/2D cell cultures to monitor membrane trafficking using readouts for autophagy induction, intracellular vacuolization, necrotic cell death, and cell and neurite migration defects that we had developed in BIOSS-1. To monitor dynamic kinase translocations we had already successfully applied the PLA technology in 2D cell cultures. Now, we will expand this analysis to organoid 3D cell culture (in collaboration with Meier/Zengerle at IMTEK) to measure multiple protein interactions in situ.
5. Statement: Due to its roles in both neurodegeneration, tumor development, aging and diabetes, SGK-1 has become a major drug target. However, individual pharmacological targeting requires screenings to manipulate selective functions of SGK-1. This proposal aims at identifying the respective screening tools for future grant applications/industry collaborations and will set the path for studying networks of disease-related gene interactions in situ on a chip (collab. with proposed project by Meier/Zengerle, Area C). With the proposed research we target research topics 1, 2, 3, 4, 6, and 8 and will develop tools for #7. We provide sensors for the analyses of technology already developed for BIOSS (PLA, microfluidics, 3D cell culture).