Spatio-temporal analysis of TGFβ receptor signalling and trafficking studied by peroxidase-mediated quantitative proximity proteomics and correlative imaging

TGF-β family proteins are central regulators of development and tissue homeostasis and the deregulation of their activities has been associated with many human diseases including cancer. TGF-β receptors are constitutively internalized via clathrin-mediated endocytosis (CME) and caveolae (lipid raft)-dependent endocytosis. Receptor endocytosis is also an important early event during TGF-β signal transduction. However, whether endocytosis is essential for TGF-β signalling remains controversial, and there are conflicting reports as to the functional contribution of CME and caveolae-dependent endocytic trafficking to TGF-β signalling output. Moreover, TGF-β signalling can occur through the canonical, SMAD-dependent pathway as well as through alternative, SMAD-independent pathways. These pathways operate in parallel and communicate with one another through cross talk, but the spatio-temporal control of this signalling network is not well understood. In addition, our molecular and mechanistic understanding of the TGF-β signalling and endocytic trafficking machinery is incomplete. Given the central role of TGF-β signalling in health and disease, detailed molecular knowledge of the pathway and its regulation is paramount.
This proposal applies a novel peroxidase-based labelling method (APEX2) to generate a comprehensive and unbiased spatio-temporal interactome of theTGF-β receptor by quantitative proximity proteomics. We will further interrogate the intracellular trafficking itinerary of the receptor by light and electron microscopy and study the functions of CME and caveolae in receptor trafficking. The combination of time-resolved proximity proteomics and high-resolution imaging will be used to produce a molecular, spatial and temporal model of TGF-β signalling and trafficking. Taken together our work will advance our mechanistic understanding of this important and versatile signalling pathway.