Eye Lens Cells Research Articles

The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293 and HELA cells. But the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1, but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Nanoceria Prevents Glucose-Induced Protein Glycation in Eye Lens Cells.

Cerium oxide nanoparticles (nanoceria) are generally known for their recyclable antioxidative properties making them an appealing biomaterial for protecting against physiological and pathological age-related changes that are caused by reactive oxygen species (ROS). Cataract is one such pathology that has been associated with oxidation and glycation of the lens proteins (crystallins) leading to aggregation and opacification. A novel coated nanoceria formulation has been previously shown to enter the human lens epithelial cells (HLECs) and protect them from oxidative stress induced by hydrogen peroxide (H2O2). In this work, the mechanism of nanoceria uptake in HLECs is studied and multiple anti-cataractogenic properties are assessed in vitro. Our results show that the nanoceria provide multiple beneficial actions to delay cataract progression by (1) acting as a catalase mimetic in cells with inhibited catalase, (2) improving reduced to oxidised glutathione ratio (GSH/GSSG) in HLECs, and (3) inhibiting the non-enzymatic glucose-induced glycation of the chaperone lens protein α-crystallin. Given the multifactorial nature of cataract progression, the varied actions of nanoceria render them promising candidates for potential non-surgical therapeutic treatment.

Qki activates Srebp2-mediated cholesterol biosynthesis for maintenance of eye lens transparency

Defective cholesterol biosynthesis in eye lens cells is often associated with cataracts; however, how genes involved in cholesterol biosynthesis are regulated in lens cells remains unclear. Here, we show that Quaking (Qki) is required for the transcriptional activation of genes involved in cholesterol biosynthesis in the eye lens. At the transcriptome level, lens-specific Qki-deficient mice present downregulation of genes associated with the cholesterol biosynthesis pathway, resulting in a significant reduction of total cholesterol level in the eye lens. Mice with Qki depletion in lens epithelium display progressive accumulation of protein aggregates, eventually leading to cataracts. Notably, these defects are attenuated by topical sterol administration. Mechanistically, we demonstrate that Qki enhances cholesterol biosynthesis by recruiting Srebp2 and Pol II in the promoter regions of cholesterol biosynthesis genes. Supporting its function as a transcription co-activator, we show that Qki directly interacts with single-stranded DNA. In conclusion, we propose that Qki-Srebp2–mediated cholesterol biosynthesis is essential for maintaining the cholesterol level that protects lens from cataract development.

Protective Role of Sphingomyelin in Eye Lens Cell Membrane Model against Oxidative Stress.

In the eye lens cell membrane, the lipid composition changes during the aging process: the proportion of sphingomyelins (SM) increases, that of phosphatidylcholines decreases. To investigate the protective role of the SMs in the lens cell membrane against oxidative damage, analytical techniques such as electrochemistry, high-resolution mass spectrometry (HR-MS), and atomic force microscopy (AFM) were applied. Supported lipid bilayers (SLB) were prepared to mimic the lens cell membrane with different fractions of PLPC/SM (PLPC: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine). The SLBs were treated with cold physical plasma. A protective effect of 30% and 44% in the presence of 25%, and 75% SM in the bilayer was observed, respectively. PLPC and SM oxidation products were determined via HR-MS for SLBs after plasma treatment. The yield of fragments gradually decreased as the SM ratio increased. Topographic images obtained by AFM of PLPC-bilayers showed SLB degradation and pore formation after plasma treatment, no degradation was observed in PLPC/SM bilayers. The results of all techniques confirm the protective role of SM in the membrane against oxidative damage and support the idea that the SM content in lens cell membrane is increased during aging in the absence of effective antioxidant systems to protect the eye from oxidative damage and to prolong lens transparency.

Myricetin Prevents Cataract Formation by Inhibiting the Apoptotic Cell Death Mediated Cataractogenesis.

BackgroundThe current research work aimed to explore the protective role of myricetin against cataractogenesis in humans, in terms of its anti-apoptotic potential.Material/MethodsHuman eye lens epithelial cells were exposed to oxidative stress by treating with hydrogen peroxide (H2O2). The levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were determined using standard detection kits. DAPI (4′,6-diamidino-2-phenylindole), AO/EB (acridine orange/ethidium bromide) and Annexin V/propidium iodide (PI) staining assays were used for the assessment of cell apoptosis. Western blotting was used to examine the protein concentrations.ResultsThe exposure of human epithelial eye lens cells to H2O2 led to significant accumulation of reactive oxygen species molecules. Treatment of the H2O2-stressed epithelial cells with myricetin caused significant (P<0.05) increased levels of SOD, CAT, and GSH. Western blot analysis also showed a significant (P<0.05) increase in the expression of SOD, CAT, and GSH levels in human epithelial eye lens cells. Additionally, myricetin administration to H2O2-treated epithelial eye lens cells caused a significant decline in cell apoptosis ratio. The induction of apoptosis was associated with upregulation of Bax and downregulation of Bcl-2.ConclusionsThe results of this study showed the potential of myricetin in protecting the apoptosis driven cataract formation in humans.

Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin D1 expression and lens shape.

Elevated cataract risk after radiation exposure was established soon after the discovery of X-rays in 1895. Today, increased cataract incidence among medical imaging practitioners and after nuclear incidents has highlighted how little is still understood about the biological responses of the lens to low-dose ionizing radiation (IR). Here, we show for the first time that in mice, lens epithelial cells (LECs) in the peripheral region repair DNA double strand breaks (DSB) after exposure to 20 and 100 mGy more slowly compared with circulating blood lymphocytes, as demonstrated by counts of γH2AX foci in cell nuclei. LECs in the central region repaired DSBs faster than either LECs in the lens periphery or lymphocytes. Although DSB markers (γH2AX, 53BP1 and RAD51) in both lens regions showed linear dose responses at the 1 h timepoint, nonlinear responses were observed in lenses for EdU (5-ethynyl-2′-deoxy-uridine) incorporation, cyclin D1 staining and cell density after 24 h at 100 and 250 mGy. After 10 months, the lens aspect ratio was also altered, an indicator of the consequences of the altered cell proliferation and cell density changes. A best-fit model demonstrated a dose-response peak at 500 mGy. These data identify specific nonlinear biological responses to low (less than 1000 mGy) dose IR-induced DNA damage in the lens epithelium.

2-Chloro-1,4-naphthoquinone derivative of quercetin as an inhibitor of aldose reductase and anti-inflammatory agent

The ability of flavonoids to affect multiple key pathways of glucose toxicity, as well as to attenuate inflammation has been well documented. In this study, the inhibition of rat lens aldose reductase by 3,7-di-hydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)-3-hydroxy-phenyl]-5-hydroxy-chromen-4-one (compound 1), was studied in greater detail in comparison with the parent quercetin (compound 2). The inhibition activity of 1, characterized by IC50 in low micromolar range, surpassed that of 2. Selectivity in relation to the closely related rat kidney aldehyde reductase was evaluated. At organ level in isolated rat lenses incubated in the presence of high glucose, compound 1 significantly inhibited accumulation of sorbitol in a concentration-dependent manner, which indicated that 1 was readily taken up by the eye lens cells and interfered with cytosolic aldose reductase. In addition, compound 1 provided macroscopic protection of colonic mucosa in experimental colitis in rats. At pharmacologically active concentrations, compound 1 and one of its potential metabolite 2-chloro-3-hydroxy-[1,4]-naphthoquinone (compound 3) did not affect osmotic fragility of red blood cells.

High-Speed Atomic Force Microscopy: Integration with Optical Microscopy and High-Speed Force Spectroscopy

High-speed atomic force microscopy (HS-AFM, [1]) offers unique novel possibilities to study single molecule dynamics [2,3]. Here we present two HS-AFM developments, first the integration of optical microscopy into HS-AFM for imaging membrane proteins on cells, and second high-speed force spectroscopy (HS-FS) for fast protein unfolding studies:HS-AFM is a powerful tool for studying structure and dynamics of proteins. So far, however, HS-AFM was restricted to well-controlled molecular systems. Here, we integrate optical microscopy (OM) into HS-AFM, allowing bright field and fluorescence microscopy, without loss of HS-AFM performance. This hybrid HS-AFM/OM setup [4] allows positioning the HS-AFM tip on an optically identified zone of interest on cells. We present movies at 960ms per frame displaying aquaporin-0 array and single molecule dynamics on intact eye lens cells. This hybrid setup allows HS-AFM imaging on cells ∼1000 times faster than conventional AFM/OM setups, and first time visualization of unlabeled membrane proteins on a eukaryotic cell under physiological conditions.The mechanical unfolding of muscle protein titin by AFM is a landmark experiment in single molecule biophysics. Molecular dynamics simulations offered an atomic level mechanistic description of the process. However, experiment and simulation differ in pulling velocity by orders of magnitude. We have developed HS-FS [5] to unfold titin at velocities reached by simulation (∼4 mm/s). Our results show that an intermediate state in a small βeta-strand pair dynamically unfolds and refolds, buffering pulling forces up to ∼100pN. Furthermore, our data indicates that the distance to the transition state of domain unfolding is much larger than previously estimated, but in better agreement with atomistic predictions.[1] Ando, et al., PNAS,2001,98(22):12468-12472.[2] Kodera, et al., Nature,2010,468(7320):72-76.[3] Casuso, et al., Nature Nanotechnology,2012,7(8):525-529.[4] Colom, et al., Nature Communications,2013,4:DOI:10.1038/ncomms3155.[5] Rico, et al., submitted,2013.

A hybrid high-speed atomic force–optical microscope for visualizing single membrane proteins on eukaryotic cells

High-speed atomic force microscopy is a powerful tool for studying structure and dynamics of proteins. So far, however, high-speed atomic force microscopy was restricted to well-controlled molecular systems of purified proteins. Here we integrate an optical microscopy path into high-speed atomic force microscopy, allowing bright field and fluorescence microscopy, without loss of high-speed atomic force microscopy performance. This hybrid high-speed atomic force microscopy/optical microscopy setup allows positioning of the high-speed atomic force microscopy tip with high spatial precision on an optically identified zone of interest on cells. We present movies at 960 ms per frame displaying aquaporin-0 array and single molecule dynamics in the plasma membrane of intact eye lens cells. This hybrid setup allows high-speed atomic force microscopy imaging on cells about 1,000 times faster than conventional atomic force microscopy/optical microscopy setups, and allows first time visualization of unlabelled membrane proteins on a eukaryotic cell under physiological conditions. This development advances high-speed atomic force microscopy from molecular to cell biology to analyse cellular processes at the membrane such as signalling, infection, transport and diffusion.

The mechanics of membrane proteins is a signature of biological function

Beyond structure, the mechanics of plasma membrane components is of key importance to biological function. Nanoscale mechanics is however poorly described due to the lack of suitable experimental tools. Here, we combined atomic force microscopy and nanomechanical mapping to analyze the structure and mechanical properties of native eye lens cell membranes. Lens membranes mainly comprise two proteins; aquaporin 0 and connexin, forming respectively thin and gap intercellular junctions that sustain mechanical stress during accommodation. Our results reveal the mechanical heterogeneity of the plasma membrane, allowing examination of the mechanical nanoenvironment of individual proteins and the flexibility of supramolecular assemblies. The remarkable rigidity of gap junctions suggests their role as stable intercellular adhesion complexes ensuring maintenance of thin junctions, which form more flexible supramolecular complexes capable of sustaining pressure differences between cells. Our work proposes the mechanical properties of individual proteins and protein domains directly related to biological function as a novel molecular signature.

Nanomechanical Characterization of the Stiffness of Eye Lens Cells: A Pilot Study

The purpose of this study is to probe the mechanical properties of individual eye lens cells isolated from nucleus and cortex of adult sheep eye lens, and to characterize the effect of cytoskeletal drugs. We used atomic force microscopy (AFM), featuring a spherical tip at the end of a soft cantilever, to indent single lens cells, and measure the Young's modulus of isolated nuclear and cortical lens cells. Measurements were performed under basal conditions, and after addition of drugs that disrupt actin filaments and microtubules. We found that single lens cells were able to maintain their shape and mechanical properties after being isolated from the lens tissue. The median Young's modulus value for nuclear lens cells (4.83 kPa) was ~ 20-fold higher than for cortical lens cells (0.22 kPa). Surprisingly, disruption of actin filaments and microtubules did not affect the measured Young's moduli. We found that single cells from the lens nucleus and cortex can be distinguished unambiguously using the elastic modulus as a criterion. The uncommon maintenance of shape and elastic properties after cell isolation together with the null effect of actin filaments and microtubules targeting drugs suggest that the mechanical stability of fiber cells is provided by cellular elements other than the usual cytoskeletal proteins.

SHSP in the eye lens: Crystallin mutations, cataract and proteostasis

α-Crystallin, a major component of the eye lens cytoplasm, is a large multimer formed from two members of the small heat shock protein (sHsp) family. Inherited crystallin mutations are a common cause of childhood cataract, whereas miscellaneous changes to the long-lived crystallins cause age-related cataract, the most common cause of blindness worldwide. Newly formed eye lens cells use proteostasis to deal with the consequences of mutations, whereas mature lens cells, devoid of the ATP-driven folding and degradation machines, are hypothesized to have the α-crystallin “holdase” chaperone function to prevent protein aggregation. We discuss the impact of truncating and missense mutations on α-crystallin, based on recent progress towards determining sHsp 3D structure. Dominant missense mutations to the “α-crystallin domain” of αA- (HSPB4) or αB-crystallin (HSPB5) occur on residues predicted to facilitate domain dynamics. αB-Crystallin is also expressed in striated muscle and mutations cause myopathy. The impact on these cellular cytoplasms is compared where sHsp multimer partners and metabolic constraints are different. Selected inherited mutations of the lens β- and γ-crystallins are considered in the context of their possible dependence on the “holdase” chaperone function of α-crystallin. Looking at discrete changes to specific crystallin polypeptide chains that can function as chaperone or substrate provide insights into the workings of a cytoplasmic proteostatic system. These observations provide a framework for validating the function of α-crystallin as a chaperone, or as a lens space filler adapted from a chaperone function. Understanding the mechanistic role of α-crystallins will aid progress in research into age-related cataract and adult-onset myopathy.This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

Impairment of Eye Lens Cell Physiology and Optics by Broadband Ultraviolet A-Ultraviolet B Radiation¶

Impairment of Eye Lens Cell Physiology and Optics by Broadband Ultraviolet A-Ultraviolet B Radiation¶

Impairment of eye lens cell physiology and optics by broadband ultraviolet A-ultraviolet B radiation.

The phototoxicity of ultraviolet A (UVA) alone and UVA plus ultraviolet B (UVB) combined on cultured porcine lenses was investigated by analyzing cellular function as measured with a fluorescence bioassay approach and optical integrity, in terms of sharpness of the lens focus as measured with a scanning laser system. The bioassay consisted of carboxyfluorescein diacetate-acetoxymethyl ester and alamarBlue fluorescent dyes. Aseptically dissected porcine lenses were maintained in modified medium 199 without phenol red supplemented with 1% penicillin-streptomycin and 4% porcine serum. At 1 week of preincubation, baseline measurements were obtained. Then the lenses were treated with single exposures of different UVA and UVB energy levels. The lenses treated with 86 J/cm2 UVA alone showed a significant (P < 0.05) decrease in cellular and optical integrity at 48 h after exposure, whereas those treated with 43 J/cm2 UVA alone did not show significant phototoxic effect. Lenses treated with 15.63 J/cm2 UVA plus 0.019 J/cm2 UVB combined showed significant adverse effects beginning from 48 h after exposure. Also, there was no recovery. These findings show that a high UVA dose alone and relatively low UVA in combination with low UVB radiant exposure can impair lens cellular and optical functions, respectively.

Coupled transcriptional and translational control of cyclin-dependent kinase inhibitor p18INK4c expression during myogenesis.

Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18INK4c gene expresses two mRNA transcripts--a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. As C2C12 cells are induced to differentiate into permanently arrested myotubes, the abundance of the p18(L) transcript decreases. The smaller p18(S) transcript expressed from a downstream promoter becomes detectable by 12 h postinduction and is the predominant transcript expressed in terminally differentiated myotubes. Both transcripts contain coding exons 2 and 3, but p18(L) uniquely contains an additional noncoding 1.2-kb exon, exon 1, corresponding exclusively to the 5' untranslated region (5' UTR). The expression pattern of the shorter p18(S) transcript, but not that of the longer p18(L) transcript, correlates with terminal differentiation of muscle, lung, liver, thymus, and eye lens cells during mouse embryo development. The presence of the long 5' UTR in exon 1 attenuated the translation of p18(L) transcript, while its absence from the shorter p18(S) transcript resulted in significantly more efficient translation of the p18 protein. Our results demonstrate that during terminal muscle cell differentiation, induction of the p18 protein is regulated by promoter switching coupled with translational control.

Polynuclear aromatic hydrocarbons and fish lens cataract: effects of benzo [a]pyrene-7,8-dihydrodiol on the macromolecular synthesis of cultured Eye cells

Eye lens cataract is common in some fish species in polycyclic aromatic hydrocarbon (PAH) heavily contaminated sites of the Elizabeth River. Chesapeake Bay, Virginia. It is not known whether these cataracts occur as a direct effect of the chemicals on the eye lens or as a manifestation of a generalized reaction at the level of the organism. For this reason, an in-vitro fish eye lens culture system has been developed to enable the examination of the direct effects of some PAH at the cellular level. Eye lens cells of the spot ( Leiostomus xanthurus) produce crystalline-like bodies under culture conditions and appear to reveal a sequence of events that resemble lens formation in vivo. The present study attempted to determine the direct effect of the breakdown product (benzo [a]pyrene-7,8-dihydrodiol) of one of the most commonly found PAH in the sites where cataract was observed, benzo[a]pyrene, on the synthesis of the major cellular macromolecules. Our results show that 7,8-diol has the ability to interfere with the synthesis of the nucleic acids DNA and RNA as well as the proteins in general. It appears, however, that the suppressive effect of the diol is more marked on DNA and RNA synthesis. The impact of these cellular aberrations on the physiological activities of the lens epithelium is presently being investigated.

Appearance of alpha-smooth muscle actin in human eye lens cells of anterior capsular cataract and in cultured bovine lens-forming cells

Abstract Using light and electron-microscopic immuno-localization techniques, and gel electrophoresis combined with immunoblotting, we have examined the expression of cytoskeletal proteins in normal human fetal, child and adult lenses, in human anterior capsular cataract and in bovine lens cells in vivo and in vitro. In this report, we focus our observations on the pattern of actin-isoform expression during normal and pathological situations in vivo and culture conditions. We have noted that cells of developing and mature human lenses as well as bovine lens cells in situ contain only β- and γ-actins. In contrast, α-smooth muscle (α-sm) actin, an isoform typical of smooth muscle differentiation, was demonstrated in bovine lens cells at different times of culture. Moreover, the multilayered cells observed in the subcapsular zone of human anterior capsular cataract were characterized by the presence of α-sm actin. Thus, extensive changes in actin-isoform expression take place in lens cells growing in culture and may also occur during cataractogenesis. The biological meaning of the appearance of a marker of myoid differentiation in the ectodermally derived lens-forming cells is discussed.

Synthesis of lens protein in vitro The lens cell-free system

1. Conditions for optimal peptide chain initiation and elongation have been determined in a cell-free system prepared from calf eye lens cells by using [ 3 5S] Met-tRNA fM et and [ 3 5S] Met-tRNA M et. 2. This cell-free system is capable of translating heterologous messenger RNA. 3. The lens proteins (crystallins), which comprise the structural matrix for the lens body, have been synthesized in the cell-free system. The amounts of newly synthesized polypeptides in vitro have been examined and compared with the proteins synthesized in organ culture. 4. The cell-free system has been employed to investigate some aspects of crystallin synthesis. We have found that synthesis of one class of protein ( β H - crystallin ) is negligible. It further appeared that differences in rate of synthesis between the polypeptide chains of α-crystallin do exist. The possible implications with respect to lens aging are discussed.

OBSERVATIONS ON THE ULTRASTRUCTURE OF THE PINEAL EYE*

SYNOPSISThe ultrastructure of most of the pineal eye cell types is surveyed, excepting the cell clusters (lens‐bodies) occurring in the lens and sometimes in the lumen. Greatly improved technique and apparatus resulted in interesting and new phase‐contrast observations, especially as regards the occurrence of ganglion cells in the nerve‐fibre layer and on the appearance of the sensory cell appendages. It is found that there are two types of sensory cells in the pineal eye; most of them are cone‐like cells very similar to the lateral eye cone cells. The pineal eye sensory cells have inner and outer segments, the former often having dense particles, mitochondria and oil droplets, and the latter, rod disks. The two segments are connected by a connecting cilium with ciliary filaments. The pineal eye sensory cells are obviously ciliary derivatives, and the possibility is discussed that they may develop from cilia seen to protrude through the microvillous border of the organ's lumen. It is further suggested that the outer segments may be transitory structures, or changeable in appearance.Structures resembling the two types of synapses described for the lateral eye are reported. The ultrastructure of the nerve‐fibre layer of the pineal eye is described.The inclusions and structure of the sensory cells and of pigment cells at various levels in the organ are described, and the nature of a coagulate which may represent a secretion or fluid occurring in the lumen is discussed. The fine structure of the pineal eye lens cells is described, and the absence of an inner limiting membrane noted.The possible functional significance of the above observations is discussed.