On the shuttling across the blood-brain barrier via tubules formation: mechanism and cargo avidity bias

The blood-brain barrier is made of polarised brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Transport across BECs is of paramount importance for nutrient uptake as well as to rid the brain of waste products. Nevertheless, currently, we do not understand how large macromolecular cargo shuttles across and how BECs discriminate between the brain-bound and own nutrients. Here, we study the low-density lipoprotein receptor-related protein 1 (LRP1) an essential regulator of BEC transport and show that it is associated with endocytic effectors, endo-lysosomal compartments as well as syndapin-2, a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily known to stabilise tubular carriers. We employed synthetic self-assembled vesicles, polymersomes, as a multivalent system with tunable avidity as a tool to investigate the mechanism of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied by biophysical modelling of transport kinetics and membrane-bound interactions. Our results demonstrate that the avidity of the ligand-receptor interaction (the overall cargo binding energy) determines the mechanism of sorting during the early stages of endocytosis and consequent trafficking. We show that high avidity cargo biases the LRP1 towards internalisation and fast degradation in BECs, while mid avidity augments the formation of syndapin-2 stabilised tubular carriers and promotes fast shuttling across BECs. Thus, we map out a very detailed mechanism where clathrin, actin, syndapin-2, dynamin and SNARE act synergistically to enable fast shuttling across BECs.