Vertebrate Lens Research Articles

Concave-to-convex curve conversion of fiber cells correlates with Y-shaped suture formation at the poles of the rodent lens

The eye lens contains convexly curved fiber cells that align in concentric layers around the lens anterior-posterior pole axis. For lens fiber differentiation at the equator, cells elongate with their apical and basal tips migrating towards the anterior and posterior poles, respectively. At each pole, the fiber tips meet opposing tips of other fiber cells, to form a suture. Although umbilical or point sutures are observed in fish and birds, line, Y- or star-shaped sutures are detected in other vertebrate lenses. Sutures that do not converge at the point are thought to result from intricate movements of the fiber tips, rather than a straightforward migration along a meridional path. The triggers that give rise to these variations are currently not understood. Our findings revealed that in the mouse embryo, the early-stage lens contains only concave curved fibers, and later, a zone of concave-to-convex curve conversion develops. At this point, a nascent suture in a linear shape appears at the posterior pole and subsequently progresses into a V-shape. This V-shape appears to further develop into a Y-shape as a branch extends from the apex of the V-shape. In lens of zebrafish and Xenopus larvae that form point sutures, this curve-conversion zone is not observed. In lens of adult birds (e.g. zebra finch) that form a point suture, these too also lack a curve-conversion zone. In our previous studies, we demonstrated that murine lens fibers undergoing curve conversion extend membrane protrusions, or lamellipodia, at their basal membranes. In line with this, we did not observe protrusions at the basal tips of fibers in the non-mammalian lenses of zebrafish, Xenopus, and zebra finch in which curve conversion does not occur. We propose that the concave-to-convex conversion in rodent lenses introduces defined paths for fiber cell tips, leading to a more elaborate and complex suture formation, compared to the simple point suture of lower vertebrates.

Characterization of βB2-crystallin tryptophan mutants reveals two different folding states in solution.

Conserved tryptophan residues are critical for the structure and the stability of β/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in β/γ-crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of β/γ-crystallin, which correlates with age-related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human βB2-crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo-oligomerization of βB2-crystallin-W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of βB2-crystallin vary over time. By using SAXS, we found that the dimer of βB2-crystallin in solution resembled the lattice βB1-crystallin dimer (face-en-face), whereas the tetramer of βB2-crystallin in solution resembled its lattice tetramer (domain-swapped). Our results suggest that homo-oligomerization of βB2-crystallin includes potential inter-subunit reactions, such as dissociation, unfolding, and re-formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of βB2-crystallin in lens.

Loss of αBa-crystallin, but not αA-crystallin, increases age-related cataract in the zebrafish lens

The vertebrate eye lens is an unusual organ in that most of its cells lack nuclei and the ability to replace aging protein. The small heat shock protein α-crystallins evolved to become key components of this lens, possibly because of their ability to prevent aggregation of aging protein that would otherwise lead to lens opacity. Most vertebrates express two α-crystallins, αA- and αB-crystallin, and mutations in each are linked to human cataract. In a mouse knockout model only the loss of αA-crystallin led to early-stage lens cataract. We have used the zebrafish as a model system to investigate the role of α-crystallins during lens development. Interestingly, while zebrafish express one lens-specific αA-crystallin gene (cryaa), they express two αB-crystallin genes, with one evolving lens specificity (cryaba) and the other retaining the broad expression of its mammalian ortholog (cryabb). In this study we used individual mutant zebrafish lines for all three α-crystallin genes to determine the impact of their loss on age-related cataract. Surprisingly, unlike mouse knockout models, we found that the loss of the αBa-crystallin gene cryaba led to an increase in lens opacity compared to cryaa null fish at 24 months of age. Loss of αA-crystallin did not increase the prevalence of cataract. We also used single cell RNA-Seq and RT-qPCR data to show a shift in the lens expression of zebrafish α-crystallins between 5 and 10 days post fertilization (dpf), with 5 and 6 dpf lenses expressing cryaa almost exclusively, and expression of cryaba and cryabb becoming more prominent after 10 dpf. These data show that cryaa is the primary α-crystallin during early lens development, while the protective role for cryaba becomes more important during lens aging. This study is the first to quantify cataract prevalence in wild-type aging zebrafish, showing that lens opacities develop in approximately 25% of fish by 18 months of age. None of the three α-crystallin mutants showed a compensatory increase in the expression of the remaining two crystallins, or in the abundant βB1-crystallin. Overall, these findings indicate an ontogenetic shift in the functional importance of individual α-crystallins during zebrafish lens development. Our finding that the lens-specific zebrafish αBa-crystallin plays the leading role in preventing age-related cataract adds a new twist to our understanding of vertebrate lens evolution.

Genetic analysis of medaka fish illuminates conserved and divergent roles of Pax6 in vertebrate eye development.

Landmark discovery of eye defects caused by Pax6 gene mutations in humans, rodents, and even fruit flies combined with Pax6 gene expression studies in various phyla, led to the master control gene hypothesis postulating that the gene is required almost universally for animal visual system development. However, this assumption has not been broadly tested in genetically trackable organisms such as vertebrates. Here, to determine the functional role of the fish orthologue of mammalian Pax6 in eye development we analyzed mutants in medaka Pax6.1 gene generated by genome editing. We found that transcription factors implicated in vertebrate lens development (Prox1a, MafB, c-Maf, FoxE3) failed to initiate expression in the presumptive lens tissue of Pax6.1 mutant fish resulting in aphakia, a phenotype observed previously in Pax6 mutant mice. Surprisingly, the overall differentiation potential of Pax6.1-deficient retinal progenitor cells (RPCs) is not severely compromised, and the only cell types affected by the absence of Pax6.1 transcription factor are retinal ganglion cells. This is in stark contrast to the situation in mice where the Pax6 gene is required cell-autonomously for the expansion of RPCs, and the differentiation of all retina cell types. Our results provide novel insight into the conserved and divergent roles of Pax6 gene orthologues in vertebrate eye development indicating that the lens-specific role is more evolutionarily conserved than the role in retina differentiation.

Animal Metabolite Database: Metabolite Concentrations in Animal Tissues and Convenient Comparison of Quantitative Metabolomic Data.

The Animal Metabolite Database (AMDB, https://amdb.online) is a freely accessible database with built-in statistical analysis tools, allowing one to browse and compare quantitative metabolomics data and raw NMR and MS data, as well as sample metadata, with a focus on the metabolite concentrations rather than on the raw data itself. AMDB also functions as a platform for the metabolomics community, providing convenient deposition and exchange of quantitative metabolomic data. To date, the majority of the data in AMDB relate to the metabolite content of the eye lens and blood of vertebrates, primarily wild species from Siberia, Russia and laboratory rodents. However, data on other tissues (muscle, heart, liver, brain, and more) are also present, and the list of species and tissues is constantly growing. Typically, every sample in AMDB contains concentrations of 60-90 of the most abundant metabolites, provided in nanomoles per gram of wet tissue weight (nmol/g). We believe that AMDB will become a widely used tool in the community, as typical metabolite baseline concentrations in tissues of animal models will aid in a wide variety of fundamental and applied scientific fields, including, but not limited to, animal modeling of human diseases, assessment of medical formulations, and evolutionary and environmental studies.

Partial assignments of tunicate βγ-crystallin structure.

Tunicate Ciona intestinalis (Ci-βγ-crystallin) is a model to study evolution of vertebrates as it has an evolutionary position between the microbial crystallins and the vertebrate lens proteins. The single-domain βγ-crystallin of the tunicate Ci-βγ-crystallin represents the single-domain ancestor of the vertebrate βγ-crystallin. It has been shown that the tunicate Ci-βγ-crystallin has a high affinity to bind to Ca2+ and that this binding greatly stabilizes the protein both thermally and chemically. However, human γS-crystallin doesn't bind to Ca2+ but it stays stable. NMR spectroscopy is used to study the structure and properties of Ci-βγ-crystallin to better understand the stability and evolutionary progression from ancestral to human lens βγ-crystallins. Residual dipolar couplings (RDCs), derived from 15N-1H in-phase/anti-phase (IPAP) heteronuclear single quantum coherence NMR spectra, provide relative orientations among internuclei vectors. These orientations can be used to build the protein backbone structure. RDCs are obtained by weakly aligning proteins in a magnetic field to create an anisotropic condition. The magnetic dipole-dipole interactions no longer average to zero and give measurable dipolar couplings. RDCs are used along with other experimental NMR data for protein structure determination. Xplor-NIH package will be used for protein structure refinement. Xplor-NIH is a popular software package for biomolecular structure determination. The program takes experimental NMR data such as RDCs and other pre-determined theoretical parameters such as covalent geometry (bond lengths and angles) to find the minimum energy structures.

Damage resistance of gamma crystallins: Weathering heat, UV, and ionizing radiation.

The γ-crystallins are structural and refractive proteins of the vertebrate lens. They belong to the βγ-crystallin superfamily, which is characterized by an exceptionally stable double-Greek key fold. In the context of the lens, these proteins must remain stable and soluble for the lifetime of the organism. Recent work in my group focuses on understanding how γ-crystallins maintain functionality in the face of damage caused by aging and oxidation. I will present results on the impact of heat, UV light, and ionizing radiation on human γS-crystallin and cataract-associated variants. We find that the γ-crystallins are not particularly resistant to damage; on the contrary, they appear to be selected for the ability to remain soluble even when the protein surface is significantly modified and transient unfolding intermediates are populated. These have been investigated using a variety of biophysical techniques, including circular dichroism, mass spectrometry, fluorescence, and NMR spectroscopy. In particular, mass spectrometry measurements on irradiated samples show evidence of both deamidation and oxidative damage, while high-resolution NMR data allow us to locate protein regions and specific residues that are involved in conformational changes, altered dynamics, and protein-protein contacts. We will discuss proposed aggregation mechanisms mediated by different types of protein modification, as well as the relevance of these findings to human cataract disease.

Structure–function relationship of α-crystallin in the context of vertebrate lens evolution and its role in eye disorders

Structure–function relationship of α-crystallin in the context of vertebrate lens evolution and its role in eye disorders

Conformational Changes of α-Crystallin Proteins Induced by Heat Stress.

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.

Pigment Epithelia of the Eye: Cell-Type Conversion in Regeneration and Disease.

Pigment epithelial cells (PECs) of the retina (RPE), ciliary body, and iris (IPE) are capable of altering their phenotype. The main pathway of phenotypic switching of eye PECs in vertebrates and humans in vivo and/or in vitro is neural/retinal. Besides, cells of amphibian IPE give rise to the lens and its derivatives, while mammalian and human RPE can be converted along the mesenchymal pathway. The PECs’ capability of conversion in vivo underlies the lens and retinal regeneration in lower vertebrates and retinal diseases such as proliferative vitreoretinopathy and fibrosis in mammals and humans. The present review considers these processes studied in vitro and in vivo in animal models and in humans. The molecular basis of conversion strategies in PECs is elucidated. Being predetermined onto- and phylogenetically, it includes a species-specific molecular context, differential expression of transcription factors, signaling pathways, and epigenomic changes. The accumulated knowledge regarding the mechanisms of PECs phenotypic switching allows the development of approaches to specified conversion for many purposes: obtaining cells for transplantation, creating conditions to stimulate natural regeneration of the retina and the lens, blocking undesirable conversions associated with eye pathology, and finding molecular markers of pathology to be targets of therapy.

Tunicate Ciona intestinalis structure analysis

Tunicate Ciona intestinalis (Ci-βγ-crystallin) is a model to study evolution of vertebrates as it has an evolutionary position between the microbial crystallins and the vertebrate lens proteins. The single-domain βγ-crystallin of the tunicate Ci-βγ-crystallin represents the single-domain ancestor of the vertebrate βγ-crystallin. It has been shown that the tunicate Ci-βγ-crystallin has a high affinity to bind to Ca2+ and that this binding greatly stabilizes the protein both thermally and chemically.

Amphibian and fish eye lens used as biomarker of remote and chronic environmental contamination

The lens of vertebrates has a general structural plan and reacts similarly to various factors of anthropogenic impact. Rainbow trout (Oncorhynchus mykiss) and grass frog (Rana temporaria L.) are the most well-studied objects of aquatic toxicology in Russia, which react to the level of exposure to toxicants in the urbanized environment and retain the consequences of this exposure. The study aims to determine the possibility of using the lens of fish and amphibians as a biomarker of chronic and long-term background environmental contamination. The fundamental importance of the work lies in the fact that for the first time the mechanisms of the response of the epithelial cells of the eye lens and lens fibers to the spectrum of various toxicants entering the environment with industrial effluents are considered. In chronic experiments, the sensitivity of the lens of Oncorhynchus mykiss fingerlings to three toxicants (dibutyl sebacate-DBS, polyterpenes-PTP, and 3-aminopropyltriethoxysilane-AGM-9) in concentrations close to the normalized valuesin the environment was studied. On adult individuals of Rana temporaria, the appearance of abnormities in the lens was studied over a long period, depending on the total environmental contamination. To assess remote environmental contamination, studies were carried out on the constantly growing lens of the grass frog, which can preserve the resulting fiber abnormities throughout its life in the form of local opacities from the harmful environmental factors impacts. On the epithelium of the lens of fish, it was found that under the influence of certain pollutants, the mitotic index (MI) in the germinal area can change over a wide range. With the combined effect of toxicants, inhibition of mitosis is also observed. Using biomicroscopy of the eye, it was possible to show that frogs from the urbanized zone of Moscow have 1.5 times more genotypic and modification changes in the lens than their relatives taken from an ecologically clean zone of the Moscow region, Mozhaisky district in the water area of the Ozerninskoye reservoir. For the first time, the sensitivity of the lens of aquatic organisms to chronic effects under experimental and background contamination of the terrestrial and aquatic environment has been shown. Therefore, taking into account the uniformity of the structure of the eye lens of fish and amphibians, as well as in other vertebrates, the lens can be used as a biomarker for assessing chronic and remote environmental contamination. The research results can be applied for a comprehensive assessment of the action of anthropogenic factors.

Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s.

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

Crystallin Proteins from Aquatic Organisms: Stability and Function in the Lens and Beyond

The crystallins are the major structural and refractive proteins of the eye lens. They are extremely long-lived proteins that maintain their stability for years or decades in the near-absence of protein turnover in the lens. In vertebrates, the structural crystallins are characterized by a common structural motif, the double-Greek key βγ-crystallin fold that originated as a Ca2+-binding protein, a function that is still maintained by microbial crystallins. Ca2+-binding βγ-crystallins are characterized by a conserved N/D-N/D-#-I-S/T-S double-clamp metal ion binding motif located in a loop region. Despite the conservation of the overall fold, the ubiquitous vertebrate structural crystallins have mostly lost their cation-binding activity in favor of stabilization via other structural means. The βγ-crystallin from the tunicate Ciona intestinalis provides a fascinating example of a protein that is intermediate in function: it tightly binds two Ca2+ ions, and is greatly stabilized by doing so. At the same time, it is expressed in the sensory organs of the larval tunicate, and earlier work by our group has shown that its refractive index increment is higher than would be expected based on its amino acid sequence alone, suggesting an additional refractive function. Structurally, the tunicate crystallin resembles one half of a vertebrate βγ-crystallin, which evolved from a common ancestor of these proteins via gene duplication and selection for new functionality in the lens. Recent work from my laboratory has focused on investigating the structure and dynamics of the tunicate crystallin in the presence and absence of Ca2+ using solution-state NMR spectroscopy. Although a crystal structure (PDB ID:2BV2) was apreviously solved by Shimeld et al., NMR data enable us to probe the molecular motions on a site-by-site basis. Identification of protein sidechains or regions of the backbone with lower-amplitude dynamics in the holo form compared to the apo form highlight features that increase the stability of the protein upon Ca2+ binding. Comparison of these sequence and structural factors with those of the vertebrate structural crystallins point to features that are needed to provide stability in the absence of cation binding as well as higher refractivity in the vertebrate lens. I will present NMR spectra and other spectroscopic data illustrating the structural and motional differences between Ca2+-bound and apo tunicate crystallin, as well as interactions with the solvent. Finally, I will discuss similarities differences between the vertebrate structural crystallins and an evolutionarily unrelated protein that serves the same function, the J2-crystallin of the box jelly Tripedalia cystophora. Unlike C. intestinalis and most other non-cephalopod invertebrates, T. cystophora has camera-type eyes with a lens and a retina. J2-crystallin has a completely different fold than the vertebrate proteins, yet it must perform the same function. I will present structural and biophysical data for this protein and discuss general functional characteristics of structural and refractive lens proteins.

Organelle degradation in the lens by PLAAT phospholipases.

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase,also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

Single cell transcriptomics of the developing zebrafish lens and identification of putative controllers of lens development

The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of β- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.

Rapamycin relieves the cataract caused by ablation of Gja8b through stimulating autophagy in zebrafish

ABSTRACT Macroautophagy/autophagy is known to be important for intracellular quality control in the lens. GJA8 is a major gap junction protein in vertebrate lenses. Mutations in GJA8 cause cataracts in humans. The well-known cataractogenesis mechanism is that mutated GJA8 leads to abnormal assembly of gap junctions, resulting in defects in intercellular communication among lens cells. In this study, we observed that ablation of Gja8b (a homolog of mammalian GJA8) in zebrafish led to severe defects in organelle degradation, an important cause of cataractogenesis in developing lens. The role of autophagy in organelle degradation in lens remains disputable. Intriguingly, we also observed that ablation of Gja8b induced deficient autophagy in the lens. More importantly, in vivo treatment of zebrafish with rapamycin, an autophagy activator that inhibits MAPK/JNK and MTORC1 signaling, stimulated autophagy in the lens and relieved the defects in organelle degradation, resulting in the mitigation of cataracts in gja8b mutant zebrafish. Conversely, inhibition of autophagy by treatment with the chemical reagent 3-MA blocked these recovery effects, suggesting the important roles of autophagy in organelle degradation in the lens in gja8b mutant zebrafish. Further studies in HLE cells revealed that GJA8 interacted with ATG proteins. Overexpression of GJA8 stimulated autophagy in HLE cells. These data suggest an unrecognized cataractogenesis mechanism caused by ablation of Gja8b and a potential treatment for cataracts by stimulating autophagy in the lens. Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; AV: autophagic vacuoles; Dpf: days post fertilization; GJA1: gap junction protein alpha 1; GJA3: gap junction protein alpha 3; GJA8: gap junction protein alpha 8; Hpf: hours post fertilization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; WT: wild type.

Connexin43 and connexin50 channels exhibit different permeability to the second messenger inositol triphosphate

Gap junction channels made of different connexins have distinct permeability to second messengers, which could affect many cell processes, including lens epithelial cell division. Here, we have compared the permeability of IP3 and Ca2+ through channels made from two connexins, Cx43 and Cx50, that are highly expressed in vertebrate lens epithelial cells. Solute transfer was measured while simultaneously monitoring junctional conductance via dual whole-cell/perforated patch clamp. HeLa cells expressing Cx43 or Cx50 were loaded with Fluo-8, and IP3 or Ca2+ were delivered via patch pipette to one cell of a pair, or to a monolayer while fluorescence intensity changes were recorded. Cx43 channels were permeable to IP3 and Ca2+. Conversely, Cx50 channels were impermeable to IP3, while exhibiting high permeation of Ca2+. Reduced Cx50 permeability to IP3 could play a role in regulating cell division and homeostasis in the lens.

Divalent Cations and the Divergence of βγ-Crystallin Function.

The βγ-crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ-crystallins bind calcium ions. The βγ-crystallin from the tunicate Ciona intestinalis (Ci-βγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-βγ is valuable for investigating the evolution of the βγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-βγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-βγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.

Constant lens fiber cell thickness in fish suggests crystallin transport to denucleated cells

The crystalline lens of the vertebrate eye grows throughout life. This growth may be enormous in fish, while the lens must be functional from larva to adult. During growth, the fiber cells of the lens must increase the concentration of specific proteins (crystallins) in the cytoplasm to increase refractive index. However, the bulk of the fiber cells in a vertebrate lens are denucleated and have no organelles to synthesize proteins. To study how this problem is solved, we first measured lens fiber cell thickness in the Nile tilapia, a teleost fish. In the lenses from 25 fish, in two size groups, fibers were considerably thinner than in other vertebrates. Fiber thickness was about constant along the radius of the lens and the same between the size groups. Since our results provided no evidence for shrinkage of lens fiber cells with growth (expected if protein concentration is increased by expelling water) we included eight additional teleost species to elucidate the mechanism by which the cells increase crystallin concentration. In all species, fiber cell thickness was about constant throughout the lens, with species-specific values. The changes in fiber cell thickness expected from an increase in crystallin concentration by removal of water were modeled. Shrinkage in cell thickness by up to 66% would have been necessary to reach the required crystallin concentration. We conclude that crystallin concentration in denucleated lens fiber cells is increased by transport of proteins from synthetically competent cells in the periphery of the lens.