A GUV-based assay to reconstitute membrane tethering in vitro.

Membrane tethering is essential for the generation of organelle contact sites, the catabolic process of autophagy, and to anchor incoming vesicles to their target membranes before vesicle fusion. Although membrane tethering is critical for cellular function, many of the current biochemical techniques to test for membrane tethering rely on indirect readouts and are limited in their ability to monitor protein localization at sites of tethering. As such, we recently developed a fluorescence microscopy-based giant unilamellar vesicle and liposome tethering assay (GLT) to study the membrane tethering properties of two autophagy proteins. In this study, we used GLT with engineered membrane tethers to demonstrate the ease of use, methods of analysis, versatility, and sensitivity of the assay. We demonstrate that: 1) GLT can be used to study liposome tethering, fusion, and phosphatase-mediated detethering of tethered liposomes, 2) GLT detects tethering with comparable sensitivity with less direct methods for monitoring membrane tethering while allowing simultaneous monitoring of membrane and protein localization, and 3) GLT can be used to monitor the kinetics of membrane tethering in real time. Collectively, our results demonstrate GLT is a broadly useful method to study membrane tethering in vitro.