Live-cell imaging of aquaporin-4 diffusion and interactions in orthogonal arrays of particles

Orthogonal arrays of particles (OAPs) have been visualized for many years by freeze-fracture electron microscopy. Our laboratory discovered that aquaporin-4 (AQP4) is the protein responsible for OAP formation by demonstrating OAPs in AQP4-transfected cells and absence of OAPs in AQP4 knockout mice. We recently developed live-cell, single-molecule imaging methods to study AQP4 diffusion and interactions in OAPs. The methods include single particle tracking of quantum-dot labeled AQP4, and total internal reflection fluorescence microscopy of green fluorescent protein (GFP) and small fluorophore-labeled AQP4. The full-length (M1) form of AQP4 diffuses freely in membranes and does not form OAPs, whereas the shorter (M23) form of AQP4 forms OAPs and is nearly immobile. Analysis of a series of AQP4 truncations, point mutants and chimeras revealed that OAP formation by AQP4-M23 is stabilized by hydrophobic tetramer-tetramer interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from blocking of this interaction by residues just upstream from Met23. These biophysical methods are being extended to identify the cellular site of AQP4 assembly, AQP4 isoform interactions, OAP size and dynamics, and the determinants of regulated OAP assembly.