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.

Orthogonal array formation by human aquaporin-4: examination of neuromyelitis optica-associated aquaporin-4 polymorphisms.

Pathogenic autoantibodies target aquaporin-4 (AQP4) water channels in individuals with neuromyelitis optica (NMO). Recently, allelic mutations were reported at residue 19 of AQP4 in three cases of NMO, and it was suggested that polymorphisms may influence disease by altering AQP4 supramolecular assembly into orthogonal arrays of particles (OAPs). We analyzed the determinants of OAP formation by human AQP4 to investigate the possible role of polymorphisms in NMO pathogenesis. NMO-associated mutations R19I and R19T in AQP4 did not affect OAP assembly, palmitoylation-dependent regulation of assembly, or NMO autoantibody binding. Residue-19 polymorphisms in AQP4 are thus unlikely to be disease relevant.

Aquaporin-4 Mz isoform: brain expression, supramolecular assembly and neuromyelitis optica antibody binding.

Water channel aquaporin-4 (AQP4) is expressed in astrocytes throughout brain and spinal cord. Two major AQP4 isoforms are expressed, M1 and M23, having different translation initiation sites. A longer isoform (Mz) has been reported in rat with translation initiation 126-bp upstream from that of M1. By immunoblot analysis of SDS and native gels probed with a C-terminus anti-AQP4 antibody, Mz was detected in rat brain as a distinct band of size ∼39 kDa. Mz was absent in human and mouse brain because of in-frame stop codons. The ability of rat Mz to form orthogonal arrays of particles (OAPs) was investigated by single particle tracking and native gel electrophoresis. We found that Mz, like M1, diffused rapidly in the cell plasma membrane and did not form OAPs. However, when co-expressed with M23, Mz associated in OAPs by forming heterotetramers with M23. Unexpectedly, Mz-expressing cells bound neuromyelitis optica autoantibodies (NMO-IgG) poorly, <5-fold compared with M1-expressing cells. Truncation analysis suggested that the poor NMO-IgG binding to Mz involves residues 31-41 upstream of Met-1. We conclude that Mz AQP4 is (a) present at low level in rat but not human or mouse brain, (b) unable to form OAPs on its own but able to associate with M23 AQP4 in heterotetramers, and (c) largely unable to bind NMO-IgG because of N-terminus effects on the structure of the AQP4/NMO-IgG binding site.

Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays.

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.

Aquaporin-4 (AQP4) associations and array dynamics probed by photobleaching and single-molecule analysis of green fluorescent protein-AQP4 chimeras.

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 "long" (M1) and "short" (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 degrees C indicated that OAPs diffuse slowly (D approximately 10(-12) cm(2)/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.

Reversible, temperature-dependent supramolecular assembly of aquaporin-4 orthogonal arrays in live cell membranes.

The shorter "M23" isoform of the glial cell water channel aquaporin-4 (AQP4) assembles into orthogonal arrays of particles (OAPs) in cell plasma membranes, whereas the full-length "M1" isoform does not. N-terminal residues are responsible for OAP formation by AQP4-M23 and for blocking of OAP formation in AQP4-M1. In investigating differences in OAP formation by certain N-terminus mutants of AQP4, as measured by freeze-fracture electron microscopy versus live-cell imaging, we discovered reversible, temperature-dependent OAP assembly of certain weakly associating AQP4 mutants. Single-particle tracking of quantum-dot-labeled AQP4 in live cells and total internal reflection fluorescence microscopy showed >80% of M23 in OAPs at 10-50 degrees C compared to <10% of M1. However, OAP formation by N-terminus cysteine-substitution mutants of M1, which probe palmitoylation-regulated OAP assembly, was strongly temperature-dependent, increasing from <10% at 37 degrees C to >70% at 10 degrees C for the double mutant M1-C13A/C17A. OAP assembly by this mutant, but not by native M23, could also be modulated by reducing its membrane density. Exposure of native M1 and single cysteine mutants to 2-bromopalmitate confirmed the presence of regulated OAP assembly by S-palmitoylation. Kinetic studies showed rapid and reversible OAP formation during cooling and OAP disassembly during heating. Our results provide what to our knowledge is the first information on the energetics of AQP4 OAP assembly in plasma membranes.

Live cell analysis of aquaporin-4 m1/m23 interactions and regulated orthogonal array assembly in glial cells.

Aquaporin-4 (AQP4) can assemble into supramolecular aggregates called orthogonal arrays of particles (OAPs). In cells expressing single AQP4 isoforms, we found previously that OAP formation by AQP4-M23 requires N terminus interactions just downstream of Met-23 and that the inability of AQP4-M1 to form OAPs involves blocking by residues upstream of Met-23. Here, we studied M1/M23 interactions and regulated OAP assembly by nanometer-resolution tracking of quantum dot-labeled AQP4 in live cells expressing differentially tagged AQP4 isoforms and in primary glial cell cultures in which native AQP4 was labeled with a monoclonal recombinant neuromyelitis optica autoantibody. OAP assembly was assessed independently by Blue Native gel electrophoresis. We found that OAPs in native glial cells could be reproduced in transfected cells expressing equal amounts of AQP4-M1 and -M23. Mutants of M23 that do not themselves form OAPs, including M23-F26Q and M23-G28P, were able to fully co-associate with native M23 to form large immobile OAPs. Analysis of a palmitoylation-null M1 mutant (C13A/C17A) indicated palmitoylation-dependent OAP assembly only in the presence of M23, with increased M1 palmitoylation causing progressive OAP disruption. Differential regulation of OAP assembly by palmitoylation, calcium elevation, and protein kinase C activation was found in primary glial cell cultures. We conclude that M1 and M23 co-assemble in AQP4 OAPs and that specific signaling events can regulate OAP assembly in glial cells.

Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging.

We investigated the molecular determinants of aquaporin-4 (AQP4) assembly in orthogonal arrays of particles (OAPs) by visualizing fluorescently labeled AQP4 mutants in cell membranes using quantum-dot single-particle tracking and total internal reflection fluorescence microscopy. The full-length ;long' (M1) form of AQP4 diffused freely in membranes and did not form OAPs, whereas the ;short' (M23) form of AQP4 formed OAPs and was nearly immobile. Analysis of AQP4 deletion mutants revealed progressive disruption of OAPs by the addition of three to seven residues at the AQP4-M23 N-terminus, with polyalanines as effective as native AQP4 fragments. OAPs disappeared upon downstream deletions of AQP4-M23, which, from analysis of point mutants, involves N-terminus interactions of residues Val24, Ala25 and Phe26. OAP formation was also prevented by introducing proline residues at sites just downstream from the hydrophobic N-terminus of AQP4-M23. AQP1, an AQP4 homolog that does not form OAPs, was induced to form OAPs upon replacement of its N-terminal domain with that of AQP4-M23. Our results indicate that OAP formation by AQP4-M23 is stabilized by hydrophobic intermolecular interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from non-selective blocking of this interaction by seven residues just upstream from Met23.

QUANTUM DOT SINGLE MOLECULE TRACKING REVEALS A WIDE RANGE OF DIFFUSIVE MOTIONS OF MEMBRANE TRANSPORT PROTEINS.

Single particle tracking (SPT) provides information about the microscopic motions of individual particles in live cells. We applied SPT to study the diffusion of membrane transport proteins in cell plasma membranes in which individual proteins are labeled with quantum dots at engineered extracellular epitopes. Software was created to deduce particle diffusive modes from quantum dot trajectories. SPT of aquaporin (AQP) water channels and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels revealed several types of diffusion. AQP1 was freely mobile in cell membranes, showing rapid, Brownian-type diffusion. The full-length (M1) isoform of AQP4 also diffused rapidly, though the diffusion of a shorter (M23) isoform of AQP4 was highly restricted due to its supermolecular assembly in raft-like orthogonal arrays. CFTR mobility was also highly restricted, in a spring-like potential, due to its tethering to the actin cytoskeleton through PDZ-domain C-terminus interactions. The biological significance of regulated diffusion of membrane transport proteins is a subject of active investigation.

Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking.

Freeze-fracture electron microscopy (FFEM) indicates that aquaporin-4 (AQP4) water channels can assemble in cell plasma membranes in orthogonal arrays of particles (OAPs). We investigated the determinants and dynamics of AQP4 assembly in OAPs by tracking single AQP4 molecules labeled with quantum dots at an engineered external epitope. In several transfected cell types, including primary astrocyte cultures, the long N-terminal "M1" form of AQP4 diffused freely, with diffusion coefficient approximately 5 x 10(-10) cm(2)/s, covering approximately 5 microm in 5 min. The short N-terminal "M23" form of AQP4, which by FFEM was found to form OAPs, was relatively immobile, moving only approximately 0.4 microm in 5 min. Actin modulation by latrunculin or jasplakinolide did not affect AQP4-M23 diffusion, but deletion of its C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding domain increased its range by approximately twofold over minutes. Biophysical analysis of short-range AQP4-M23 diffusion within OAPs indicated a spring-like potential, with a restoring force of approximately 6.5 pN/microm. These and additional experiments indicated that 1) AQP4-M1 and AQP4-M23 isoforms do not coassociate in OAPs; 2) OAPs can be imaged directly by total internal reflection fluorescence microscopy; and 3) OAPs are relatively fixed, noninterconvertible assemblies that do not require cytoskeletal or PDZ-mediated interactions for formation. Our measurements are the first to visualize OAPs in live cells.