Nanoscale regulation of small GTPase switches at the trans-Golgi network/recycling endosomal interface
Cell homeostasis, differentiation and development rely on the delivery of important receptors and signaling molecules to their correct intracellular and plasma membrane destinations. The trans-Golgi network (TGN) and recycling endosomal compartments (RE) are two major sorting stations along the secretory pathway in polarized epithelial cells. Our understanding of how they orchestrate sorting and exchange material/cargoes has been hampered by their close co-localization in the perinuclear area and the impossibility to image their dynamic relationship with diffraction-limited light microscopy. Additionally, while individual machinery compo- nents involved in sorting at the TGN/RE interface have been efficiently catalogued and their interactions mapped, understanding the dynamics and the spatio-temporal organization of proteins has been neglected. Molecular switches of the ARF (ADP ribosylation factor) family of GTPases are major players in the orchestration of trafficking at the Golgi. While their role in formation of COPI vesicles is well established, their function in polarized secretion and beyond sorting at the Golgi is unclear. A systematic study that investigates the nanoscale organization and dynamics of these molecular components at the TGN/RE interface and their role in sorting of cargoes and mediating communication between the closely juxtaposed TGN and RE compart- ments in polarized epithelial cells is lacking. Harnessing CRISPR/Cas9 knock-ins (KIs) in combination with live-cell super-resolution imaging, functional trafficking assays as well as quantitative mass spectrometry- based proteomics we will dissect the localization, function and regulation of small GTPase-based molecular switches within the TGN/RE network to facilitate the segregation and polarized sorting of cargoes in epithelial cells.