Determining the Spatial Relationship of Membrane-Bound Aquaporin-4 Autoantibodies by STED Nanoscopy.

Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for superresolution nanoscopy. The organization of aquaporin-4 (AQP4) into large plasma membrane assemblies provides an opportunity to image membrane-bound AQP4 antibodies (AQP4-IgG) and evaluate changes in their spatial distribution due to alterations in AQP4 isoform expression and AQP4-IgG epitope specificity. Using stimulated emission depletion nanoscopy, we imaged secondary antibody labeling of monoclonal AQP4-IgGs with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4). Imaging secondary antibodies bound to M1-AQP4 allowed us to infer the size of individual AQP4-IgG binding events. This information was used to model the assembly of larger AQP4-IgG complexes on M23-AQP4 arrays. A scoring algorithm was generated from these models to characterize the spatial arrangement of bound AQP4-IgG antibodies, yielding multiple epitope-specific patterns of bound antibodies on M23-AQP4 arrays. Our results delineate an approach to infer spatial relationships within protein arrays using stimulated emission depletion nanoscopy, offering insight into how information on single antibody fluorescence events can be used to extract information from dense protein assemblies under a biologic context.

Three-dimensional location of the main immunogenic region of the acetylcholine receptor.

About two-thirds of the antibodies to the nicotinic acetylcholine (ACh) receptor in patients with the autoimmune disease myasthenia gravis bind to the main immunogenic region (MIR). This is small, well-defined region on each of the two alpha subunits, containing residues 67-76 (alpha 67-76). By determining the structure of the ACh receptor complexed with two different fragments of an MIR-directed antibody, we have determined the three-dimensional location of the MIR (and therefore residues alpha 67-76) to be at the extreme synaptic end of each alpha subunit. The antibody fragments extend from the binding site away from the receptor axis and into the synaptic cleft, minimizing any steric interference neighboring ACh receptors might have with their binding.

Visualization of natural autoantibody polyreactivity by rotary metal-shadowing electron microscopy.

Immune complexes formed by mouse polyspecific natural autoantibodies and various structurally different antigens, such as DNA, tubulin and myosin, were analysed by rotary-shadowing electron microscopy. Each of the four natural IgM autoantibodies studied (E7, D23, 3C3 and M2-9) recognized multiple epitopes on the myosin molecule. These results, confirmed by immunoblotting experiments using myosin subfragments as antigens, strikingly contrasted with those obtained with an induced myosin-specific IgG antibody which interacted with a single myosin antigenic site. Based on the measurements of the antibody position on the antigen, made on a series of electron micrographs, two negatively charged myosin peptides were prepared by solid phase synthesis. Polymeric forms of one of the two peptides interacted with the positively charged CDR part of E7 and inhibited the binding of E7 and M2-9 to myosin. The importance of charge in the observed cross-reactivities was further supported by enzyme immunoassays showing that most, but not all, antigen/natural autoantibody interactions were sensitive to increasing concentrations of NaCl.