TRACE · Resolving the spacial distribution of TGN subdomains at super-resolution and deciphering their role in specific cargo sorting

The trans-Golgi network (TGN) serves as a central sorting station of membrane traffic standing at the intersection of secretory and endocytic pathways, a feature that is now proposed to be a common trait between plant, yeast and animal cells. The multi-identity of TGN relies on its partitioning in subdomains but, due to the lack of resolution in conventional microscopy, it is still poorly understood how TGN subdomains relate dynamically to each other, or acquire their identity, and how they achieve the sorting of a variety of cargo proteins to different destinations. The applicant previously identified of at least two subdomains, one labeled by the R-SNARE VAMP721 and the coat protein AP-1 and that is involved in secretory trafficking, and another labeled by VAMP727 and AP-4 and that is involved in vacuolar trafficking. The host lab previously demonstrated by lipidomic analyses that sphingolipids with very long chain fatty acid were enriched in Q-SNARE SYP61-labelled TGN compartments and were critical for polar sorting of the auxin carrier PIN2 to apical plasma membrane. The goal of this project is now to join forces, expertise and scientific networks to gain from each other and tackle the challenging questions of: 1) what is the exact structure of TGN, how are TGN subdomains spatially arranged between each other and how do they behave dynamically, this will be addressed by super-resolution microscopy, 2) what is the proteo-lipidic composition of TGN subdomains and what is the role of lipids in TGN partitioning, 3) how the identified subdomains are functionally important for cargo sorting and trafficking to different destination by employing a novel pulse-chase method. This project is timely, realistic, focused and represents a unique combination of expertise from the applicant in super-resolution 3D live imaging of TGN subdomains and the host lab in TGN-mediated trafficking, lipid analytical biochemistry and super-resolution imaging.