The structure of the cytoplasm of lens fibers as determined by conical tomography

Abstract

Studies using conventional electron microscopy describe the cytoplasm of lens fiber cells as having essentially an amorphous structure. We hypothesized that significant structural detail might have been lost as a result of projecting the entire thickness of the section (50–100 nm) onto a single plane (the “projection artifact”). To test this hypothesis, we studied the 3D-structure of rat lens cortical fibers before and after extracting the “soluble” crystallins with low ionic strength buffers to make “ghosts.” Tomographic series in conical geometry were collected at 55° tilts and by 5° rotations until completing a 360° turn by low dose methods. They were aligned using fiduciary points, reconstructed with the weighted back projection algorithm and refined by projection matching. Analysis of the 3D-maps included semiautomatic density segmentation using a computer program based on the watershed algorithm. We found that the cytoplasm of cortical fibers, though appearing amorphous in regions of the highest density, was in fact comprised of an ordered structure resembling a “clustered matrix.” The matrix was comprised of thin (~6 nm diameter) filaments bent sharply at 110–120° angles and studded with cubeshaped particles (the “beaded” filaments). In cortical fibers, the particles measured a = 14 ± 2, b = 13 ± 2 and c = 10 ± 2.4 nm (n = 30, mean ± SD) and were spaced at distances measuring 27.5 ± 2.4 nm apart (n = 8, mean ± SD), center-to-center. The matrix was formed as “beaded” filaments, bound to clusters of “soluble” proteins, crossed each other at nearly perpendicular angles. The matrix also made contact with the plasma membrane at a large number of distinct regions. We thus concluded that the cytoplasm of cortical lens fibers is comprised of a cytoskeletal matrix of “beaded” filaments that organize the “soluble” crystallins in separate regions. The association of this matrix with the plasma membrane allows the lens to maintain its structural integrity, while its association with crystallins yields its long-term transparency. Loss of either function likely would play a significant role in cataract formation.

Electron tomography of fiber cell cytoplasm and dense cores of multilamellar bodies from human age-related nuclear cataracts

“…4B, inset arrows), revealing the distinctive single bilayer membrane with the same curvature and overall thickness along its length. Because both halves of the bilayer are visible, it is reasonable to estimate the resolution of the images and reconstructions at about 3–4 nm, similar to the estimates given in other tomography studies of thin sections (Burette et al, 2012; Schietroma et al, 2009). These images also illustrate some important advantages of tomographic reconstruction.…”

“…Because of the distinctive spherical shape and high concentration of alpha-crystallin, it was expected that the particles would be readily recognizable in intact cytoplasm. However, this does not appear to be the case, even for young rat lens cortical cytoplasm that was analyzed by conical tomography in which the high density of protein obscured the underlying fine structure of the cytoplasmic components (Schietroma et al, 2009; Zampighi et al, 2011). Furthermore, the aging of the lens induces numerous modifications to alpha-crystallin based on recent proteomics analyses (Asomugha et al, 2010; Grey and Schey, 2009; Sharma and Santhoshkumar, 2009; Su et al, 2011).…”

“…Many of the ultrastructural alterations can be visualized with thin-section transmission electron microscopy (TEM), although technical limitations, due to artifacts of projecting structural features onto the image plane, have put serious constraints on the information available from morphological studies. To overcome some of these limitations, we have used electron tomography (Frey et al, 2006; Schietroma et al, 2009), a valuable tool that helps visualize the 3D arrangement of lens fiber cell proteins in the cytoplasm and particle cores. The advantages offered by tomography include improvement in image quality by potentially minimizing image degradation caused by the superimposition of mass within a thin section.…”

Ultrastructural analysis of the human lens fiber cell remodeling zone and the initiation of cellular compaction

“…However, beaded filaments and other cytoskeletal elements are not normally visible in thin sections of intact fiber cells and have only been visualized after the soluble crystallins have been removed leaving a cytoplasmic ghost (Alizadeh et al, 2004; Schietroma et al, 2009). It is well established that the beaded filament proteins are degraded with age, which may be caused by endogenous enzymes (Sandilands et al, 1995; Blankenship et al, 2001; FitzGerald, 2009).…”

The Three-Dimensional Distribution of αA-Crystalline in Rat Lenses and Its Possible Relation to Transparency

“…These pentalamellar structures represent regions where the plasma membranes of neighboring fiber cells form the “gap junctions,” the organelles that function in lens cell-to-cell communication [25], [26]. In contrast, the cytoplasm of these developed fibers appeared “amorphous” or unstructured with no indication of the ordered “beaded” filaments identified in fractions isolated from lens tissues [20], [22].…”