During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers1. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect2–4, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin5,6. Clathrin-independent endocytic events are often less reliant on dynamin7, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we found that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which both are clathrin-independent endocytic cargoes8. In controlled in vitro systems, endoA2 reshapes membranes prior to scission. Furthermore, we demonstrate that endoA2, dynamin, and actin contribute in parallel to the scission of Shiga toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings finally highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling force-driven dynamic scission.
Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.
Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be achieved by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real time imaging of three different endocytic ligands - folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single particle tracking of Gal3 or STxB-functionalized, QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs that bear a unique stoichiometry of endocytic ligands represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics.
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