Organizing Large GTPase Atlastin-Mediated Membrane Fusion by Programmable DNA Origami Platforms

In eukaryotic cells, the endoplasmic reticulum (ER) utilizes membrane fusion to form interconnected networks. GTP hydrolysis-triggered conformational changes in ER-anchored atlastins (ATLs) have been shown to be responsible for this process, which is facilitated by the amphipathic helix within its C-terminal tail (AH_ATL-C). However, how the multiple ATL molecules cooperate to complete membrane tethering and subsequent fusion is still not known. Here, we used a programmable DNA origami ring to generate uniformly sized proteoliposomes containing a controlled maximum number of Drosophila ATL (dmATL) molecules. Using this system, we demonstrated at the single-event level that one pair of dmATL molecules is capable of mediating membrane tethering but not lipid mixing. To investigate the subsequent fusion event, we developed programmable DNA soccer-ball frameworks as a template for the generation of tethered liposomes. With a combination of molecular dynamics simulations and DNA-ring-templated single-vesicle fusion assays, we found that the AH_ATL-C of dmATL is sufficient to promote the merging of closely apposed membranes by enhancing bilayer fluctuations under specific lipid composition condition. Our results provide mechanistic insights into dmATL-mediated homotypic membrane fusion, and our adaptable DNA nanostructures offer powerful tools for studying other biological processes requiring predetermined numbers of molecules or involving tethered membranes.