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Guido A. Zampighi, Catherine Cheng, Xiaohua Gong, Lorenzo Zampighi; How a Mutation in the αA-Crystalline Causes Cataracts in Mice: A Molecular Explanation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2279.
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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.
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.
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.
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.
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