Biological glass: structural determinants of eye lens transparency

Abstract

The purpose of the lens is to project a sharply focused, undistorted image of the visual surround onto the neural retina. The first pre-requisite, therefore, is that the tissue should be transparent. Despite the presence of remarkably high levels of protein, the lens cytosol remains transparent as a result of short-range-order interactions between the proteins. At a cellular level, the programmed elimination of nuclei and other light-scattering organelles from cells located within the pupillary space contributes directly to tissue transparency. Scattering at the cell borders is minimized by the close apposition of lens fibre cells facilitated by a plethora of adhesive proteins, some expressed only in the lens. Similarly, refractive index matching between lens membranes and cytosol is believed to minimize scatter. Refractive index matching between the cytoplasm of adjacent cells is achieved through the formation of cellular fusions that allow the intermingling of proteins. Together, these structural adaptations serve to minimize light scatter and enable this living, cellular structure to function as 'biological glass'.

Figure 1.

Cellular organization of the vertebrate lens. The lens is bounded by the lens capsule (Cap). (a,b) Its anterior surface is covered by a monolayer of epithelial cells (Ep). The primary lens fibre cells (pLF) are formed early in embryonic development and constitute the embryonic nucleus in the adult lens. Secondary lens fibre cells are formed continuously by mitosis of cells in the germinative zone (GZ) at the equatorial margin of the epithelium. The fibres are stacked one upon another in meridional rows (c). These secondary lens fibre cells differentiate (b, dLF) and subsequently elongate (b, eLF) beneath the apical surface of the epithelium (f, aEp) and along the posterior capsule until their tips reach the anterior (aS) and posterior (pS) sutures. Mature fibre cells in the organelle-free zone (OFZ) of the lens lack nuclei and other organelles. Most fibre cells are hexagonal in cross-section and very regularly organized in closed sheets as shown in the light and scanning electron microscope images of (d) and (e). Diagram adapted from Shi et al. [9]. GC, growth cone.

Figure 2.

Cellular architecture of the vertebrate lens. Scanning electron micrographs of the lenses of zebrafish (a,b), rabbit and (c,d), human lenses. The inset in (c) shows the complex branching pattern of sutures in the adult human lens. ant, anterior pole; post, posterior pole; eq, equator.

Figure 3.

Three-dimensional structure of mouse lens cells at various stages of differentiation as revealed by confocal microscopy. (a,b). Lens epithelial cells showing (a) basal or (b) apical surfaces. (c) Young elongating fibre cells located near the surface of a two-month-old mouse lens. The fibres are initially smooth and ribbon-like. Their membrane surface features a large number of gap junction plaques (green) visualized here by immunofluorescence with anti-connexin (Cx) 50. (d) At this stage, the fibre cell is in the process of losing its organelles. At the membrane surface, ball-and-socket processes (enriched with Cx50) are formed on the broad face of the lateral membrane (arrow). Smaller, finger-like structures protrude from the narrow membrane faces (arrowheads). (e) With the disappearance of organelles the fibres take on an undulating appearance. (f–i). Fibre cells dissected from progressively deeper cell layers. The primary fibre cells from the centre of the lens (i) are characterized by a very irregular structure. The approximate location (distance beneath the lens equatorial surface) of the fibre cells is indicated in parentheses. Mouse lenses of this age (two months) are approximately 1800 µm in diameter.

Figure 4.

Membrane associations between fibre cells. (a,b) Scanning EM images of interlocking edge protrusions (asterisks and white and black arrows) between superficial lens fibres. In deeper cortical regions (c,d) the edge protrusions (arrowheads) become tortuous and ball and sockets junctions (black and white arrows) appear. Freeze fracture images at low (e) magnification support the close interlocking of neighbouring fibres by edge protrusions (arrows) as seen in scanning EM images (a,b). High magnification images (f) reveal the relatively small number of particles on the protoplasmic (p–f) and external (e–f) faces of the fibre membranes. Incidentally a gap junction is found (GJ). High magnification freeze fracture images (g) show the high density of membrane particles on ball-and-socket junctions (inset). This is corroborated by low (inset) and high magnification TEM observations (h) suggesting that these junctions are giant gap junctions with the typical close apposition of adjacent membranes. In freeze fracture images of deep cortical regions (i), lens fibre membranes show grooves and ridges (microplicae) on their surface which correspond to undulating membranes of neighbouring fibres as shown in TEM images (j). Gap junctions (arrowed in i) are found occasionally on these membranes. Note the high spatial order of the cytoplasm in (j). Figure (a–d) are taken from a rabbit lens; (e–l) from a human lens. cyt, fibre cytoplasm.

Figure 5.

Fibre cell denucleation in rodent lenses. (a) Programmed degradation of nuclei occurs in cortical lens fibre cells of rats raised under normal conditions. (b) Chromatin breakdown is inhibited in rats fed a tryptophan-deficient diet. (c) Transient reintroduction of tryptophan causes chromatin breakdown in fibres that underwent denucleation during the period tryptophan was present. (d) Electron micrograph of a nucleus from the cortex of a tryptophan-deficient lens. Note the segregation of chromatin/DNA (electron dense) from other nuclear constituents. Inset shows a light micrograph of the same nucleus stained with Hoechst 33258 dye to visualize DNA. (e) At higher magnification, small (approx. 10 nm) particles (putative proteasomes) are visible in the matrix of disintegrating nuclei tryptophan-deficient rats. (f) Similar condensed chromatin/DNA is also found in normal rat lenses and in young human lenses. (g) Denucleation also occurs in the cortex of wild-type mouse lenses but is blocked in lenses from DNase-IIβ-null mice (h). Ep, epithelium; OFZ, organelle-free zone.

Figure 6.

Calpain activation in cells bordering the OFZ in a lens from a three-day-old mouse. The distribution of calpain-cleaved spectrin (green) is used as a surrogate for calpain activation. Calpain-cleaved spectrin (arrow) first appears in fibre cells undergoing nuclear degeneration. Lens cell nuclei are visualized with propidium iodide (red). Image adapted from De Maria et al. [34].

Figure 7.

Disaggregation of lens fibre cells in the Lim2-null mouse lens. Partially-fixed lenses were teased apart with fine forceps. Note that in the absence of Lim2, fibre cells readily separate into individual cells. These data support the notion that Lim2 may have an adhesive function in the lens.

Figure 8.

Refractive index matching of lens membranes and cytoplasm.

Figure 9.

Lim2-dependent cell fusion during mouse lens fibre cell differentiation. (a–c) A rotational series of volumetric reconstructions of a region of the outer cortex of a wild-type lens. GFP expression was induced in scattered lens cells. In the outer region (depths less than 50 µm) GFP is retained in the cytoplasm of expressing cells. Three such cells are shown. At greater depths, the cytoplasm of neighbouring cells becomes continuously linked through regions of partial membrane fusion. As a result, discretely labelled cells are not observed in the deeper cell layers. Rather, GFP diffuses among a cluster of neighbouring fibre cells (asterisks). The formation of cellular fusions is dependent on the presence of an intrinsic membrane protein, Lim2 (a.k.a. MP20). In wild-type mouse lenses (d) GFP is restricted to the cytoplasm of fibre cells located near the lens surface (arrow) but diffuses from expressing cells when those cells are buried to a depth of more than 50 µm (arrowhead). In the absence of Lim2 (e), fusions are not formed and GFP is retained by both superficial fibre cells (arrow) and fibre cells located deep below the lens surface (arrowhead). Scale bars, (a,b,c) 150 µm; (d,e) 250 µm.