Abstract
Purpose: :
To profile the 3D-distribution of the αA-crystalline in wild type controls and to determine how a single amino acid substitution (αA-Y118D) induces a dominant cataract in lenses of mice and humans.
Methods: :
Thin sectioning electron microscopy was performed in lenses from: a) wild type mice labeled with anti-αA-crystalline and 2nm and 5nm diameter gold particles, b) wild type unlabeled, and c) mice carrying the αA-Y118D mutation in the absence of αB-crystalline. Tomograms calculated from conical series (55° tilts and 5° rotations) were reconstructed using the weighted back projection algorithm and refined by projection matching. Analysis of the tomograms relied on density segmentation methods based on the watershed transformation.
Results: :
Reconstructions of fiber cells of wild-type mice labeled with antibody/gold particle conjugates indicated that αA-crystalline monomers or dimers decorate thin cytoskeleton filaments spaced ~7.5nm center-to-center apart. The "decorated" filaments intersected to form ~16nm diameter "bead" assemblies or larger ~32nm diameter particles. In fibers carrying the αA-Y118D mutation, the densities of "decorated" filaments and "beads" decreased steeply when normalized per unit volume of cytoplasm. In contrast, the density of larger diameter particles increased by a factor of four with respect to wild type controls. Additional changes included: a) the precipitation of soluble proteins in ordered platelets and b) the fragmentation of the plasma membrane and the communication of the fiber cytoplasm with the extra-cellular space.
Conclusions: :
Analysis of the 3D-structure of fiber cells indicates that the αA-Y118D mutation changes the aggregation state of the αA-crystalline from monomers/dimers decorating thin filaments to particles comprised of larger number of subunits. In addition, other soluble crystallines formed ordered thin platelets in the fiber cytoplasm. We conclude that the αA-Y118D mutation in the αA-crystalline causes cataracts by a gain-of-order mechanism instead of the formation of light-scattering protein aggregates.
Keywords: cataract • image processing • cytoskeleton