AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.     

The pulling,pushing and fusing of lens fibers

Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.     

Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry,Transparency, and Mechanical Stiffness of the Mouse Lens

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.     

Changes in fibronectin staining in the human lens during ageing and cataractogenesis

The content and localization of fibronectin, an extracellular glycoprotein, in the serial sections of lenses of normal human donors and cataractous patients of different ages were determined by the indirect immunoperoxidase staining technique. This was followed by the evaluation with quantitative morphometric analysis. It was shown that fibronectin was present in the area of cell contacts as single deposits of faint orange-brown stained material in the lens samples of young donors. The fibronectin level was raised in lens sections from aged donors. Its accumulation was detected mostly within the spaces of the lens fiber cells. At different stages of cataractogenesis a dramatic decrease of the fibronectin content was detected in the lens sections obtained from patients of different ages. A new linear spectrophotometric technique was developed for evaluation of the lens transparency, to correlate the lens opacity with corresponding histological data obtained from the immunostaining technique. Morphological studies performed further suggested that the lens fiber cell plasma membrane structures were deteriorated. This was observed as changes of fibronectin staining in the lens sections at different periods of human ageing and cataract development. It is concluded that a decrease of fibronectin staining in the human lens is an indication for the structural damage of the lens fiber cell plasma membranes during ageing and cataractogenesis.     

Ras signaling is essential for lens cell proliferation and lens growth during development

The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of beta- and gamma-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 A, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 Å, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

Connexins in Lens Development and Cataractogenesis

The lens is an avascular organ that transmits and focuses light images onto the retina. Intercellular gap junction channels, formed by at least three different connexin protein subunits, α1 (connexin43 or Gja1), α3 (connexin46 or Gja3) and α8 (connexin50 or Gja8), are utilized to transport metabolites, ions and water in the lens. In combination with physiological and biochemical analyses, recent genetic studies have significantly improved our understanding about the roles of diverse gap junction channels formed by α3 and α8 connexin subunits during lens development and cataract formation. These studies have demonstrated that α3 connexin is essential for lens transparency while α8 connexin is important for lens growth and transparency. Diverse gap junction channels formed by α3 and α8 subunits are important for the differentiation, elongation and maturation of lens fiber cells. Aberrant gap junction communication, caused by alterations of channel assembly, channel gating or channel conductance, can lead to different types of cataracts. These findings provide some molecular insights for essential roles of connexins and gap junctions in lens formation and the establishment and maintenance of lifelong lens transparency.     

Inhibition of alpha-crystallin aggregation by gamma-crystallin

The transparency of the mammalian lens is primarily maintained by short range order among the major proteins of the lens fiber cells, the crystallins. Although these proteins are highly conserved at the amino acid sequence level, it has proven difficult to establish that they possess other than structural functions. We find that when non-lens proteins are added to concentrated solutions of alpha-crystallin, aggregation is induced, presumably through excluded volume effects. In contrast, the monomeric gamma-crystallins and the low molecular weight form of beta-crystallin (beta L) cause a decrease in the size of alpha-crystallin. When the naturally aggregated form of alpha-crystallin is examined, gamma- and beta L-crystallin, as well as a reducing agent, also cause partial dissociation as detected by dynamic light scattering and size exclusion chromatography, while no effect is seen with non-crystallin proteins. Furthermore, the chemical cross-linking of alpha-crystallin is inhibited by gamma- and beta L-crystallin but not by other proteins. The ability of gamma-crystallin to inhibit the association of alpha-crystallin is primarily localized to the gamma-II form which contains a high degree of exposed thiols. Only small amounts of gamma- and beta L-crystallin, however, can be cross-linked to alpha-crystallin in mixtures of the three proteins even at very high protein concentrations. These results suggest that one possible role for the lower molecular weight crystallins may be to minimize through a reductive effect the intrinsic tendency of alpha-crystallin to aggregate, an association reaction implicated in the loss of lens transparency.     

Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration

Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0^+/−) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0^+/−/AQP1^+/−) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS–PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28 kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26–24 kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (P_f) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA. Transmission and scanning electron micrographs of lenses of both mouse models showed increased extracellular space between fiber cells. Water content determination study showed increase in water in the lenses of these mouse models. In summary, lens transparency, CTCA and compact packing of fiber cells were affected due to the loss of 50% AQP0 leading to larger extracellular space, more water content and SA, possibly due to alteration in RING. To our knowledge, this is the first report identifying the role of AQP0 in RING development to ward off lens SA during focusing.     

-Crystallin quaternary structure and interactive properties control eye lens transparency

The eye lens is the foremost biological system where function is directly under control of the physico-chemical properties of the cytoplasmic macromolecular solution. Indeed, lens transparency and opacity, lens refractive index gradient and viscosity, are the result of the structural and interactive properties of the crystallins, of their stability, of the fine tuning of their interaction potentials and associations at different levels of organization. Among the different crystallin classes, -crystallins have represented a major challenge for a long time. The -crystallin secondary, tertiary and quaternary structures are still unknown. On the functional side, however, it is established that -crystallin quaternary structure and repulsive interactions determine lens transparency, whereas the -crystallin chaperone effect most probably plays a role in the aging process. In the present paper, we recall the physico-chemical properties and the quaternary structure features of -crystallins that were demonstrated to control light scattering and transparency. The interest of a crystallin mixture for lens function is discussed. Then, a formal approach is proposed to design models for the -crystallin quaternary structure, including the question of whether -crystallins assemble with symmetry. An hypothesis relevant to the fold of the -crystallin C-terminal domain is presented in another paper in this issue.     

Ageing and vision: structure, stability and function of lens crystallins

The -, β- and γ-crystallins are the major protein components of the vertebrate eye lens, -crystallin as a molecular chaperone as well as a structural protein, β- and γ-crystallins as structural proteins. For the lens to be able to retain life-long transparency in the absence of protein turnover, the crystallins must meet not only the requirement of solubility associated with high cellular concentration but that of longevity as well. For proteins, longevity is commonly assumed to be correlated with long-term retention of native structure, which in turn can be due to inherent thermodynamic stability, efficient capture and refolding of non-native protein by chaperones, or a combination of both. Understanding how the specific interactions that confer intrinsic stability of the protein fold are combined with the stabilizing effect of protein assembly, and how the non-specific interactions and associations of the assemblies enable the generation of highly concentrated solutions, is thus of importance to understand the loss of transparency of the lens with age. Post-translational modification can have a major effect on protein stability but an emerging theme of the few studies of the effect of post-translational modification of the crystallins is one of solubility and assembly. Here we review the structure, assembly, interactions, stability and post-translational modifications of the crystallins, not only in isolation but also as part of a multi-component system. The available data are discussed in the context of the establishment, the maintenance and finally, with age, the loss of transparency of the lens. Understanding the structural basis of protein stability and interactions in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.     

Molecular basis of eye lens transparency. Osmotic pressure and X-ray analysis of alpha-crystallin solutions

Short range, liquid-like order of the crystallin proteins accounts for eye lens transparency. The relationship between structural and thermodynamic properties of eye lens was further investigated using osmotic pressure and small-angle X-ray scattering measurements of calf lens alpha-crystallins. The consistency of both data sets confirms that the macroscopic thermodynamic properties are determined by the structural properties accessible to X-ray scattering. In addition, the experimental data were correctly accounted for using a model developed in liquid-state physics: the rescaled mean spherical approximation combined with a Verwey-Overbeek potential. This model provides as best fit parameters the excluded volume, the charge and the diameter of an "equivalent" particle that compare well with the corresponding values found in the literature for alpha-crystallins. As a result, transparency may now be expressed as a function of a few structural parameters, the role of which is discussed. The approach presented here may be extended to studies of the thermodynamic-structural relationships of other protein solutions.     

Human cataract: the mechanisms responsible; light and butterfly eyes

Age-related cataract is the leading cause of world blindness. Until recently, the biochemical mechanisms that result in human cataract formation have remained a mystery. In the case of nuclear cataract, it is becoming apparent that changes that take place within the lens at middle age may be ultimately responsible. The centre of the lens contains proteins that were synthesised prior to birth and while these crystallins are remarkably stable, it appears that an antioxidant environment may be necessary in order for them to remain soluble and for lens transparency. Once an internal barrier to the movement of small molecules, such as antioxidants, develops in the normal lens at middle age, the long-lived proteins in the lens centre become susceptible both to covalent attachment of reactive molecules, such as UV filters, and to oxidation. These processes of protein modification may, over time, lead inevitably to lens opacification and cataract.     

Evolution of eye lens crystallins: the stress connection

Crystallins, the structural proteins of the eye lens, ensure the transparency and integrity of the lens throughout life. Recent sequence comparisons have shown that evolution has recruited crystallins among already existing heat-shock proteins and stress-inducible enzymes.     

Effects of dietary deficiency of selective amino acids on the function of the cornea and lens in rats

Summary.  Effects of dietary deficiencies of tryptophan and methionin on the transparency of cornea and lens were investigated in young rats (Brown-Norway, BN; Sprague-Dawley, SD) over 3 months. Transparency of the cornea and lens were evaluated in weekly intervals using a photo-slitlamp microscope. After sacrifice and lens fresh weight determination the lenses were prepared for histopathology. Methionin deficiency had no effect on the parameters investigated. Tryptophan deficiency caused severe loss of body weight in both strains, with additional loss of hair in SD rats. These developed corneal neovascularisations and cataracts. BN rats showed an enhanced zone of discontinuity in the lens. Diet intermission arrested the pathological processes in the eye which restarted when feeding the diet again. This observation is supported by lens fresh weight data. DNA staining evidenced that tryptophan deficiency arrested lens fiber maturation in both strains but stimulated corneal neovascularisation only in SD rats. Received June 29, 2001 Accepted August 6, 2001 Published online July 31, 2002     

A signaling role for the uncleaved form of alpha 6 integrin in differentiating lens fiber cells

Many alpha integrin subunits are cleaved during their processing to yield heavy and light chains, which remain associated by disulfide bonds. While uncleaved alpha integrin subunits can form functional receptors that sometimes have distinct signaling roles from their better-characterized endoproteolytically cleaved counterparts, their expression at the cell surface and their association with signaling complexes have yet to be determined in vivo. In this study, we demonstrate that, in differentiating lens fiber cells, the uncleaved form of alpha 6 integrin was expressed at the cell surface. This form of alpha 6 integrin coimmunoprecipitated with both the signaling adaptor molecule Shc and its downstream effector Grb2, suggesting that, in lens fiber cells, uncleaved alpha 6 integrin was associated with a Shc-mediated signaling complex. We show that expression of the cleaved form of alpha 6 integrin progressively decreased relative to uncleaved alpha 6 integrin as the state of lens cell differentiation increased, resulting in the predominance of uncleaved alpha 6 integrin in the lens fiber cell zones. Interestingly, we previously have shown that alpha 6 integrin is localized principally along the extensive cell-cell interfaces of these lens fiber cells, in the absence of its extracellular matrix ligand laminin. While we found that the cleaved form of alpha 6 integrin contained both high mannose and complex sugars, the uncleaved form of alpha 6 integrin contained only high mannose sugars. These properties suggest that the uncleaved form of alpha 6 integrin may have a unique role in the embryonic lens. Its high association with Shc and Grb2 in the differentiating cortical fiber cell zone indicates that alpha 6 integrin may provide a cell survival signal in the presence of the apoptotic-like processes that are initiated in this region of the embryonic lens to clear the lens cells of their organelles.     

Corneal crystallins and the development of cellular transparency

Past studies have established that the cornea like the lens abundantly expresses a few water-soluble enzyme/proteins in a taxon specific fashion. Based on these similarities it has been proposed that the lens and the cornea form a structural unit, the 'refracton', that has co-evolved through gene sharing to maximize light transmission and refraction to the retina. Thus far, the analogy between corneal crystallins and lens crystallins has been limited to similarities in the abundant expression, with few reports concerning their structural function. This review covers recent studies that establish a clear relationship between expression of corneal crystallins and light scattering from corneal stromal cells, i.e. keratocytes, that support a structural role for corneal crystallins in the development of transparency similar to that of lens crystallins that would be consistent with the 'refracton' hypothesis.     

Expression of c-myc protooncogene in rat lens cells during development,maturation and reversal of galactose cataracts

It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate. Elevated levels of c-myc mRNA have usually been correlated with rapid cell growth and increased entry of cells into the S phase. Therefore, changes in c-myc mRNA levels may provide an early indication of the stimulation of lens epithelial cells to proliferate and differentiate, which has been postulated to be an early and important event in response to lens cell injury by galactose. By Northern blot hybridization analysis we quantitated c-myc mRNA levels in the lens capsule epithelia of rats (1) exposed to galactose, and (2) undergoing a partial recovery from the galactose-induced cell damage. At the onset of lens cell damage, we find c-myc mRNA to elevate to 6-fold by 24 hr, and by 48 hr decreases to about 3-fold the normal levels. During recovery, c-myc mRNA continues to be expressed at high levels approaching a 10-fold increase by day 12, then decreasing to levels of about 8-fold the control by day 30. The 24 h transitory elevation in c-myc mRNA in lens epithelial cells is in accord with our previous observations on the 24 h increase in MP26, crystallin and aldose reductase mRNAs following a high influx of galactose. Therefore, the elevation in c-myc mRNA as well suggest that galactose appears to cause lens cells to undergo an early transitory period of gene induction following the exposure of lens cells to galactose.