Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes

Abstract

Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can co-mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ∼20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm2 with decreasing M1∶M23 ratio from 1∶1 to 1∶3, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs of area 2142±829 nm2, which increased to 5137±1119 nm2 with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average D ∼ 10−12 cm2/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs.