Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes.
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 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 a primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm(2) 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 with an area of 2142±829 nm(2), which increased to 5137±1119 nm(2) with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average ~10(-12) cm(2)/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.
Department of Medicine, University of California, San Francisco, CA 94143,  USA.
Andrea
A
Rossi
Tobias J
TJ
Moritz
Julien
J
Ratelade
A S
AS
Verkman
D000818:Animals; D051401:Aquaporin 4 / Q000737:chemistry* / Q000378:metabolism*; D001253:Astrocytes / Q000378:metabolism; D016466:CHO Cells; D016923:Cell Death; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D016000:Cluster Analysis; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms / Q000378:metabolism; D020836:Protein Structure, Quaternary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D051401:Aquaporin 4; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins