Orientation microscopy and cryo-electron tomography to study glycan-dependent assembly of membrane nanodomains – OMEGA-MEMDO

The existence of membrane nanodomains is accepted but the structural principles according to which proteins and lipids are co-assembled and the mechanisms by which membrane nanodomain construction affords biological outcomes remain poorly explored.

At the cell surface, glycans provide a link for glycolipid and glycoprotein co-assembly into membrane nanodomains from which tubular endocytic pits emerge for the generation of clathrin-independent endocytic carriers. Oligomeric glycan-binding proteins, termed galectins, act as the molecular “glue”. We hypothesize that glycoprotein a5b1 integrin nucleates and corrals a core of galectin-3 (Gal3) oligomers onto membranes that drive endocytic membrane bending.

With the OMEGA-MEMDO program, we use forefront techniques to monitor the membrane nanodomain construction process from cellular to molecular scales. Oligomerization site mutants of Gal3 and glycosylation site mutants of a5b1 integrin will be tested on cells by lattice light sheet microscopy for endocytic uptake, and by Single Molecule Orientation-Localization Microscopy (SMOLM) for orientation and order of lipids and proteins at the nanoscale. Purified Gal3, a5b1 integrin, and mutants will be analyzed by cryo-electron tomography (cryo-ET) on model membrane systems to visualize individual proteins and their conformation, spatial organization, and membrane environment. Developments in image processing and artificial intelligence will be performed to extract and correlate cellular and molecular information from SMOLM and cryo-ET datasets.

Our interdisciplinary network unites cellular, chemical, and structural biologists, physicists, and biomathematicians to unravel the structural principles according to which glycan-dependent nanodomains are built to tune the cell surface dynamics of critical signaling receptors. Since most proteins carry glycans at the cell surface, these nanodomains have the potential to impact any biological function that originates from there.