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.     

Evidences for a Leaky Scanning Mechanism for the Synthesis of the Shorter M23 Protein Isoform of Aquaporin-4: IMPLICATION IN ORTHOGONAL ARRAY FORMATION AND NEUROMYELITIS OPTICA ANTIBODY INTERACTION*

Aquaporin-4 (AQP4) exists as two major isoforms that differ in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which can further aggregate in the plasma membrane to form typical orthogonal arrays of particles (OAPs) whose dimension depends on the ratio of the M1 and M23. In this study, we tested the hypothesis that the M23 isoform can be produced directly by the M1 mRNA. In cells transiently transfected with AQP4-M1 coding sequence we observed besides AQP4-M1 the additional presence of the AQP4-M23 isoform associated with the formation of typical OAPs observable by two-dimensional blue native/SDS-PAGE and total internal reflection microscopy. The mutation of the second in-frame methionine M23 in AQP4-M1 (AQP4-M1^M23I) prevented the expression of the M23 isoform and the formation of OAPs. We propose “leaky scanning” as a translational mechanism for the expression of AQP4-M23 protein isoform and that the formation of OAPs may occur even in the absence of AQP4-M23 mRNA. This mechanism can have important pathophysiological implications for the cell regulation of the M1/M23 ratio and thus OAP size. In this study we also provide evidence that AQP4-M1 is mobile in the plasma membrane, that it is inserted and not excluded into immobile OAPs, and that it is an important determinant of OAP structure and size.     

Model of aquaporin-4 supramolecular assembly in orthogonal arrays based on heterotetrameric association of M1-M23 isoforms

Tetramers of aquaporin-4 (AQP4) water channels form supramolecular assemblies in cell membranes called orthogonal arrays of particles (OAPs). We previously reported evidence that a short (M23) AQP4 isoform produced by alternative splicing forms OAPs by an intermolecular N-terminus interaction, whereas the full-length (M1) AQP4 isoform does not by itself form OAPs but can coassemble with M23 in OAPs as heterotetramers. Here, we developed a model to predict number distributions of OAP size, shape, and composition as a function M23:M1 molar ratio. Model specifications included: random tetrameric assembly of M1 with M23; intertetramer associations between M23 and M23, but not between M1 and M23 or M1; and a free energy constraint limiting OAP size. Model predictions were tested by total internal reflection fluorescence microscopy of AQP4-green-fluorescent protein chimeras and native gel electrophoresis of cells expressing different M23:M1 ratios. Experimentally validated model predictions included: 1), greatly increased OAP size with increasing M23:M1 ratio; 2), marked heterogeneity in OAP size at fixed M23:M1, with increased M23 fraction in larger OAPs; and 3), preferential M1 localization at the periphery of OAPs. The model was also applied to test predictions about binding to AQP4 OAPs of a pathogenic AQP4 autoantibody found in the neuroinflammatory demyelinating disease neuromyelitis optica. Our model of AQP4 OAPs links a molecular-level interaction of AQP4 with its supramolecular assembly in cell membranes.     

Post-Golgi supramolecular assembly of aquaporin-4 in orthogonal arrays

The supramolecular assembly of aquaporin-4 (AQP4) in orthogonal arrays of particles (OAPs) involves N-terminus interactions of the M23-AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4-transfected cells, (ii) photobleaching recovery of green fluorescent protein-AQP4 chimeras in live cells and (iii) freeze-fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes, but not in the Golgi, was not related to AQP4 density, pH, membrane lipid composition, C-terminal PDZ domain interactions or α-syntrophin expression. Remarkably, however, fusion of AQP4-containing Golgi vesicles with (AQP4-free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature-dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to ～110 nm diameter, but not to ～220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post-Golgi phenomenon involving plasma membrane-specific factor(s). Post-Golgi and membrane curvature-dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4-facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica.     

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 °C indicated that OAPs diffuse slowly (D ∼ 10⁻¹² cm²/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.     

Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which binding of pathogenic autoantibodies (NMO-IgG) to astrocyte aquaporin-4 (AQP4) causes complement-dependent cytotoxicity (CDC) and inflammation. We previously reported a wide range of binding affinities of NMO-IgGs to AQP4 in separate tetramers versus intramembrane aggregates (orthogonal arrays of particles, OAPs). We report here a second, independent mechanism by which CDC is affected by AQP4 assembly. Utilizing lactate dehydrogenase release and live/dead cell cytotoxicity assays, we found in different cell lines, and with different monoclonal and patient-derived NMO-IgGs, that CDC was greatly (>100-fold) reduced in cells expressing M1- versus M23-AQP4. Studies using a M23-AQP4 mutant containing an OAP-disrupting mutation, and in cells expressing AQP4 in different M1/M23 ratios, indicated that NMO-IgG-dependent CDC requires AQP4 OAP assembly. In contrast, antibody-dependent cell-mediated cytotoxicity produced by natural killer cells did not depend on AQP4 OAP assembly. Measurements of C1q binding and complement attack complex (C9neo) supported the conclusion that the greatly enhanced CDC by OAPs is due to efficient, multivalent binding of C1q to clustered NMO-IgG on OAPs. We conclude that AQP4 assembly in OAPs is required for CDC in NMO, establishing a new mechanism of OAP-dependent NMO pathogenesis. Disruption of AQP4 OAPs may greatly reduce NMO-IgG dependent CDC and NMO pathology.     

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.     

Formation of aquaporin-4 arrays is inhibited by palmitoylation of N-terminal cysteine residues

Tetramers of the mammalian water channel aquaporin-4 (AQP4) assemble into square arrays and mediate bidirectional water transport across the blood-brain interface. The aqp4 gene expresses two splicing isoforms. Only the shorter AQP4M23 isoform assembles into square arrays, while the longer AQP4M1 isoform interferes with array formation, presumably due to the additional 22 N-terminal residues. To understand why the N-terminus of AQP4M1 interferes with array formation, we constructed a series of N-terminal deletion mutants and examined their ability to form square arrays in Chinese hamster ovary (CHO) cells using SDS-digested freeze fracture replica labeling. Mutants with deletions of less than seventeen N-terminal residues did not form square arrays and showed dispersed immunogold labels against AQP4 molecules, whereas more deletions led to the formation of square arrays labeled with immunogolds. Furthermore, mutagenic substitution of the two cysteine residues at the position 13 and 17 in the N-terminus of AQP4M1 also resulted in the square array formation. Biochemical analysis and metabolic labeling of transfected CHO cells revealed that the two N-terminal cysteines of AQP4M1 are palmitoylated. These results suggest that palmitoylation of the N-terminal cysteines is the reason for the inability of AQP4M1 to form square arrays.     

Formation of aquaporin-4 arrays is inhibited by palmitoylation of N-terminal cysteine residues

Tetramers of the mammalian water channel aquaporin-4 (AQP4) assemble into square arrays and mediate bidirectional water transport across the blood-brain interface. The aqp4 gene expresses two splicing isoforms. Only the shorter AQP4M23 isoform assembles into square arrays, while the longer AQP4M1 isoform interferes with array formation, presumably due to the additional 22 N-terminal residues. To understand why the N-terminus of AQP4M1 interferes with array formation, we constructed a series of N-terminal deletion mutants and examined their ability to form square arrays in Chinese hamster ovary (CHO) cells using SDS-digested freeze fracture replica labeling. Mutants with deletions of less than seventeen N-terminal residues did not form square arrays and showed dispersed immunogold labels against AQP4 molecules, whereas more deletions led to the formation of square arrays labeled with immunogolds. Furthermore, mutagenic substitution of the two cysteine residues at the position 13 and 17 in the N-terminus of AQP4M1 also resulted in the square array formation. Biochemical analysis and metabolic labeling of transfected CHO cells revealed that the two N-terminal cysteines of AQP4M1 are palmitoylated. These results suggest that palmitoylation of the N-terminal cysteines is the reason for the inability of AQP4M1 to form square arrays.     

Aquaporin-4 Autoantibodies in Neuromyelitis Optica: AQP4 Isoform-Dependent Sensitivity and Specificity

Neuromyelitis Optica (NMO) is an autoimmune demyelinating disease, characterized by the presence of autoantibody (NMO-IgG) to Aquaporin-4 (AQP4). NMO-IgG identification supports NMO diagnosis and several diagnostic tests have been developed, but their sensitivity is too variable, and some assay show low sensitivity. This impairs correct diagnosis of NMO. By cell based assay (CBA) we here evaluate the efficacy of different strategies to express AQP4 in mammalian cells in terms of: a) AQP4 translation initiation signals; b) AQP4 isoforms (M1 and M23) and fluorescent tag position; c) NMO serum concentration and AQP4 degradation. Our results demonstrate that when using AQP4-M1, the nucleotide in position −3 of the AUG greatly affects the AQP4-M1/M23 protein ratio, NMO-IgG binding, and consequently test sensitivity. Test sensitivity was highest with M23 expressing cells (97.5%) and only 27.5% with AQP4-M1. The fluorescent tag added to the N-terminus of AQP4-M23 considerably affected the NMO-IgG binding, and test sensitivity, due to disruption of AQP4 suprastructures. Furthermore, sera used at high concentration resulted in AQP4 degradation which affected test sensitivity. To further evaluate the reliability of the M23 based CBA test, samples of one NMO patient collected during about 2 years clinical follow-up were tested. The results of serum titer correlated with disease activity and treatment response. In conclusion, we provide a molecular explanation for the contrasting CBA test data reported and suggest the use of M23 with a C-terminus fluorescent tag as the proper test for NMO diagnosis.     

Heterotetrameric composition of aquaporin-4 water channels.

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.     

Development of an Aquaporin-4 Orthogonal Array of Particle-Based ELISA for Neuromyelitis Optica Autoantibodies Detection

Serological markers of Nuromyelitis Optica (NMO), an autoimmune disorder of the central nervous system, are autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4). We have previously demonstrated that the main epitopes for these autoantibodies (AQP4-IgG) are generated by the supramolecular arrangement of AQP4 tetramers into an Orthogonal Array of Particles (OAPs). Many tests have been developed to detect AQP4-IgG in patient sera but several procedural issues affect OAP assembly and consequently test sensitivity. To date, the protein based ELISA test shows the lowest sensitivity while representing a valid alternative to the more sensitive cell based assay (CBA), which, however, shows economic, technical and interpretation problems. Here we have developed a high perfomance ELISA in which native OAPs are used as the molecular target. To this aim a native size exclusion chromatography method has been developed to isolate integral, highly pure and AQP4-IgG-recognized OAPs from rat brain. These OAPs were immobilized and oriented on a plastic plate by a sandwich approach and 139 human sera were tested, including 67 sera from NMO patients. The OAP-ELISA showed a 99% specificity and a higher sensitivity (91%) compared to the CBA test. A comparative analysis revealed an end-point titer three orders of magnitude higher than the commercial ELISA and six times higher than our in-house CBA test. We show that CNS-extracted OAPs are crucial elements in order to perform an efficient AQP4-IgG test and the OAP-ELISA developed represents a valid alternative to the CBA currently used.     

Histamine treatment induces rearrangements of orthogonal arrays of particles (OAPs) in human AQP4-expressing gastric cells

To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 +/- 0.3 x 10-4 to 16.37 +/- 0.5 x 10-4 cm/s at 20 degrees C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 +/- 0.3% under basal condition and decreased by 50% to 1.2 +/- 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pf decreased by approximately 35% in 20-min histamine-stimulated cells. Both Pf and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.     

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp⁶⁹) of TM2 for NMO-IgG epitope assembly. Mutation of Asp⁶⁹ to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp⁶⁹ mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D⁶⁹H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp⁶⁹ is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.     

An allograft glioma model reveals the dependence of aquaporin-4 expression on the brain microenvironment

Aquaporin-4 (AQP4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. In contrast, most cultivated glioma cell lines don't express AQP4, and primary cell cultures of human glioblastoma lose it during the first passages. Accordingly, in C6 cells and RG2 cells, two glioma cell lines of the rat, and in SMA mouse glioma cell lines, we found no AQP4 expression. We confirmed an AQP4 loss in primary human glioblastoma cell cultures after a few passages. RG-2 glioma cells if grafted into the brain developed AQP4 expression. This led us consider the possibility of AQP4 expression depends on brain microenvironment. In previous studies, we observed that the typical morphological conformation of AQP4 as orthogonal arrays of particles (OAP) depended on the extracellular matrix component agrin. In this study, we showed for the first time implanted AQP4 negative glioma cells in animal brain or flank to express AQP4 specifically in the intracerebral gliomas but neither in the extracranial nor in the flank gliomas. AQP4 expression in intracerebral gliomas went along with an OAP loss, compared to normal brain tissue. AQP4 staining in vivo normally is polarized in the astrocytic endfoot membranes at the glia limitans superficialis and perivascularis, but in C6 and RG2 tumors the AQP4 staining is redistributed over the whole glioma cell as in human glioblastoma. In contrast, primary rat or mouse astrocytes in culture did not lose their ability to express AQP4, and they were able to form few OAPs.     

Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.     

The Pathway of Assembly of Intermediate Filaments from Recombinant α-Internexin

The pathway of filament assembly from the neuronal intermediate filament α-intermexin was investigated. Optimal assembly occurred in solutions of pH 6.5 to 7 and moderate ionic strength at 37°C. Short filaments formed upon dialysis at 24°C, which elongated further when incubated at 37°C. Soluble forms of α-internexin were characterized by analytical ultracentrifugation and electron microscopy. In 10 mM Tris, pH 8, conditions that favor formation of tetramers and other small oligomers for other intermediate filament proteins, α-internexin formed 10.5 S particles, apparently unit-length half-filaments in the form of rods 10.6 nm in diameter and 68 nm long. Dialysis vs the same buffer with added 10 mM NaCl yielded 16 S rods, probably unit-length filaments, of the same length but 13.0 nm in diameter. At 50 mM NaCl, rods about 13 nm in diameter and heterogeneous in length were observed in electron micrographs, apparently formed from longitudinal annealing of unit-length rods. The results favor a model of assembly in which coiled coil dimers aggregate laterally to form first “unit-length half-filaments” (Herrmann, H., and Aebi, U. (1998)Curr. Opin. Struct. Biol.8, 177–185) and then “unit-length filaments,” which subsequently elongate by annealing.