Live-cell imaging of aquaporin-4 supramolecular assembly and diffusion

Aquaporin-4 (AQP4) is a water channel expressed in astrocytes throughout the central nervous system, as well as in epithelial cells in various peripheral organs. AQP4 is involved in brain water balance, neuroexcitation, astrocyte migration, and neuroinflammation and is the target of pathogenic autoantibodies in neuromyelitis optica. Two AQP4 isoforms produced by alternative splicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular aggregates called orthogonal arrays of particles (OAPs). OAPs have been studied morphologically, by freeze-fracture electron microscopy, and biochemically, by native gel electrophoresis. We have applied single-molecule and high-resolution fluorescence microscopy methods to visualize AQP4 and OAPs in live cells. Quantum dot single particle tracking of fluorescently labeled AQP4 has quantified AQP4 diffusion in membranes, and has elucidated the molecular determinants and regulation of OAP formation. The composition, structure, and kinetics of OAPs containing fluorescent protein-AQP4 chimeras have been studied utilizing total internal reflection fluorescence microscopy, single-molecule photobleaching, and super-resolution imaging methods. The biophysical data afforded by live-cell imaging of AQP4 and OAPs has provided new insights in the roles of AQP4 in organ physiology and neurological disease.