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Guido A. Zampighi, Salvatore Lanzavecchia, Lorenzo Zampighi; Molecular Immunocytochemistry: A Method to Study the Changes Induced by Mutations of the αA-crystalline in Lens Fiber Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1616.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the feasibility of applying the newly developed method of molecular immunocytochemistry to profile the 3D-distribution of the αA-crystalline protein in wild type and mutated mice lenses. Molecular immunocytochemistry is based on conical tomography and allows studying proteins in cells at high resolution (~2nm) and in three-dimensions.
We collected conical series at 55° tilt and 5° rotation increments of fiber cells from lenses: a) labeled with anti-αA and 2nm and 5nm gold particle conjugates, b) unlabeled and c) carrying a mutation in the protein where a tyrosine was replaced by aspartate in position 118 (Y118D). The conical series were reconstructed using the weighted back projection algorithm and refined using a strategy based on projection matching. Analysis of the gold particle maps provided Euclidean distances, dihedral angles and polygons created by the 3D-distribution of the αA-crystalline in the cytoplasm. Analysis of the "reference space" provided the envelope structure of the assemblies containing the αA-crystalline (the "prototypes"). The information is used as constraints to profile the αA-crystalline in lenses carrying the Y118D mutation.
For each of the 325 gold particles, we measured: the volume, the peak intensity and the x,y,z coordinates of the mass centers. From these measurements, we determined the Euclidean distances between nearest particles. Histograms of distances between 5nm gold particles were centered on two Gaussian at 14nm (HWHM=2.0nm) and 24nm (HWHM=3.5nm). Histograms of the distances between 2nm gold particles were centered on a single Gaussian at 7.5nm (HWHM=3.6nm). Analysis of the "reference space" defined tethers (~12nm in length) linking the gold particles to the assemblies containing the αA-crystalline. These assemblies appeared as: a) thin filaments decorated with granules of αA-crystalline spaced 6-7nm center-to-center apart and b) ~15nm diameter "beads." The Y118D mutation of the αA-crystalline alters the dimensions of the granules decorating the filaments and association of "beads" with the plasma membrane.
The high spatial resolution (~2nm) and the capacity of profiling an unlimited number of proteins are unequaled properties of molecular immunocytochemistry for determining changes induced by physiological and pathological conditions in the mammalian lens.
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