Abstract
Lens transparency depends on the accumulation of massive quantities (600–800 mg/ml) of twelve primary crystallines and two truncated crystallines in highly elongated “fiber” cells. Despite numerous studies, major unanswered questions are how this heterogeneous group of proteins becomes organized to bestow the lens with its unique optical properties and how it changes during cataract formation. Using novel methods based on conical tomography and labeling with antibody/gold conjugates, we have profiled the 3D-distribution of the αA-crystalline in rat lenses at ∼2 nm resolutions and three-dimensions. Analysis of tomograms calculated from lenses labeled with anti-αA-crystalline and gold particles (∼3 nm and ∼7 nm diameter) revealed geometric patterns shaped as lines, isosceles triangles and polyhedrons. A Gaussian distribution centered at ∼7.5 nm fitted the distances between the ∼3 nm diameter gold conjugates. A Gaussian distribution centered at ∼14 nm fitted the Euclidian distances between the smaller and the larger gold particles and another Gaussian at 21–24 nm the distances between the larger particles. Independent of their diameters, tethers of 14–17 nm in length connected files of gold particles to thin filaments or clusters to ∼15 nm diameter “beads.” We used the information gathered from tomograms of labeled lenses to determine the distribution of the αA-crystalline in unlabeled lenses. We found that αA-crystalline monomers spaced ∼7 nm or αA-crystalline dimers spaced ∼15 nm center-to-center apart decorated thin filaments of the lens cytoskeleton. It thus seems likely that lost or gain of long-range order determines the 3D-structure of the fiber cell and possible also cataract formation.
Highlights
To attain transparency, the lens underwent a series of evolutionary adaptations that include the elimination of blood vessels from its interior and the accumulation of massive quantities (600–800 mg/ml) of a heterogeneous group of small molecular weight (20–30 kDa) proteins, called crystallines, in the cytoplasm of highly elongated fiber cells [1,2,3,4]
Evidence supporting long-range order includes measurement of the Euclidian distances and the geometric patterns adopted by the aA-crystalline in tomograms calculated from labeled and unlabeled fiber cells (Tables 1&2)
The single line pattern reflects the monomers or the dimers of the aA-crystalline repeating alongside intermediate filaments
Summary
The lens underwent a series of evolutionary adaptations that include the elimination of blood vessels from its interior and the accumulation of massive quantities (600–800 mg/ml) of a heterogeneous group of small molecular weight (20–30 kDa) proteins, called crystallines, in the cytoplasm of highly elongated fiber cells [1,2,3,4]. A large body of experimental evidence suggests that crystallines form multi-subunit assemblies that are organized with ‘‘shortrange’’ order of dense solutions in the cytoplasm of fiber cells [9,10,11,12]. Evidence suggesting this organization includes: a) the ‘‘amorphous’’ structure of the cytoplasm of fiber cells observed in conventional electron microscopy studies [13,14,15,16], and b) the absence of long-range order observed in solutions of purified crystallines [17,18,19]. Studies of fractions isolated from chick and later mammalian lenses reveal a unique type of protein assembly, called the ‘‘beaded’’ filament, which is difficult to reconcile with the short-range order of dense solutions
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