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Hunger and thirst have distinct goals but control similar ingestive behaviors, and little is known about neural processes that are shared between these behavioral states. We identify glutamatergic neurons in the peri-locus coeruleus (periLCVGLUT2 neurons) as a polysynaptic convergence node from separate energy-sensitive and hydration-sensitive cell populations. We develop methods for stable hindbrain calcium imaging in free-moving mice, which show that periLCVGLUT2 neurons are tuned to ingestive behaviors and respond similarly to food or water consumption. PeriLCVGLUT2 neurons are scalably inhibited by palatability and homeostatic need during consumption. Inhibition of periLCVGLUT2 neurons is rewarding and increases consumption by enhancing palatability and prolonging ingestion duration. These properties comprise a double-negative feedback relationship that sustains food or water consumption without affecting food- or water-seeking. PeriLCVGLUT2 neurons are a hub between hunger and thirst that specifically controls motivation for food and water ingestion, which is a factor that contributes to hedonic overeating and obesity.
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Regulación del Apetito/fisiología , Ingestión de Líquidos/fisiología , Ingestión de Alimentos/fisiología , Locus Coeruleus/citología , Red Nerviosa/fisiología , Neuronas/fisiología , Rombencéfalo/fisiología , Análisis de la Célula Individual/métodos , Animales , Apetito/fisiología , Escala de Evaluación de la Conducta , Retroalimentación , Conducta Alimentaria/fisiología , Femenino , Glutamina/metabolismo , Glutamina/fisiología , Homeostasis/fisiología , Hambre/fisiología , Masculino , Ratones , Ratones Noqueados , Motivación/fisiología , Neuronas/efectos de los fármacos , Proteínas Recombinantes , Recompensa , Rombencéfalo/citología , Rombencéfalo/diagnóstico por imagen , Gusto/fisiología , Sed/fisiologíaRESUMEN
The spheroidal shape of the eye lens is crucial for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that, in the mouse lens, membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating that lamellipodium formation is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Rac-dependent protrusions, suggesting changes in the activity of FGF controlling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin and actin reduced the height of both early and later fibers, indicating that elongation of these fibers relies on actomyosin contractility. Consistent with this, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose that it does so through regulation of Rho activity.
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Factores de Crecimiento de Fibroblastos , Cristalino , Ratones , Animales , Cristalino/metabolismo , Epitelio/metabolismo , Actinas/metabolismo , Diferenciación Celular/fisiologíaRESUMEN
Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataracts. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq and CUT&RUN-seq to discover previously unreported mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Furthermore, we identify an epigenetic paradigm of cellular differentiation, defined by progressive loss of the H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation.
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Catarata , Cristalino , Humanos , Multiómica , Catarata/genética , Diferenciación Celular/genética , OjoRESUMEN
Seismically imaged axial melt lenses (AMLs) are seen almost everywhere along the axis of fast-spreading ridges but at only a few localized segment centers on slow-spreading ridges. Standard models assuming that AMLs form when melt percolating upward pools where freezing produces an impermeable cap do not explain this fundamental observation. To tackle this long-standing problem, we combine a crustal density model and a thermal model with a recent mechanical model for sill formation. The mechanical model predicts that AMLs form below the axial lithosphere but only if the average density of the axial brittle lithosphere is not greater than the magma density. For standard thermal models, crustal density structures inferred from seismic velocity data and normal crustal thicknesses, AMLs are found to be stable along all of a ridge segment for spreading rates greater than about 50 mm/y. To explain slow-spreading observations, we assume that a share of the melt produced by the mantle upwelling all along a segment is focused to the segment center. Some of this melt partially crystallizes, releasing latent heat, before the evolved magma flows along the axis to build the crust away from the segment center. This "extra" heat, beyond what is supplied by the magma that builds the crust near the segment center, results in the lithosphere thin enough for stable melt lenses at the segment center. Our results are consistent with observations and offer a quantitative explanation of the marked difference in the distribution of AMLs along fast- versus slow-spreading centers.
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Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90ß (HSP90ß) plays a fundamental role in suppressing cataractogenesis. HSP90ß is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90ß is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90ß silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90ß can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90ß silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90ß and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90ß is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.
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Catarata , Proteínas HSP90 de Choque Térmico , Proteína p53 Supresora de Tumor , Animales , Humanos , Ratones , Envejecimiento/genética , Catarata/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Cuerpos Multivesiculares/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. Here, we show that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Our transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisona , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles, yet maintains transparency for at least 5 decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is in addition hypothesized that transparency is maintained by a microcirculation system that delivers antioxidants to the lens nucleus and exports small molecule waste. Common data-dependent acquisition methods are hindered by dynamic range of lens protein expression and provide limited context to age-related changes in the lens. In this study, we utilized data-independent acquisition mass spectrometry to analyze the urea-insoluble membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens microcirculation system and oxidative stress response. In this pilot cohort, we measured 4788 distinct protein groups, 46,681 peptides, and 7592 deamidated sequences, more than in any previous human lens data-dependent acquisition approach. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell-cell contacts while retaining networks related to oxidative stress response. Furthermore, we identified multiple antioxidant transporter proteins not previously detected in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that aquaporin-5, among other proteins, is modified with age by post-translational modifications including deamidation and truncation. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decreased fiber cell permeability and result in ARNC formation.
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Catarata , Cristalino , Humanos , Proteoma/metabolismo , Cristalino/química , Cristalino/metabolismo , Catarata/metabolismo , Antioxidantes/metabolismoRESUMEN
Crystallins comprise the protein-rich tissue of the eye lens. Of the three most common vertebrate subtypes, ß-crystallins exhibit the widest degree of polydispersity due to their complex multimerization properties in situ. While polydispersity enables precise packing densities across the concentration gradient of the lens for vision, it is unclear why there is such a high degree of structural complexity within the ß-crystallin subtype and what the role of this feature is in the lens. To investigate this, we first characterized ß-crystallin polydispersity and then established a method to dynamically disrupt it in a process that is dependent on isoform composition and the presence of divalent cationic salts (CaCl2 or MgCl2). We used size-exclusion chromatography together with dynamic light scattering and mass spectrometry to show how high concentrations of divalent cations dissociate ß-crystallin oligomers, reduce polydispersity, and shift the overall protein surface charge-properties that can be reversed when salts are removed. While the direct, physiological relevance of these divalent cations in the lens is still under investigation, our results support that specific isoforms of ß-crystallin modulate polydispersity through multiple chemical equilibria and that this native state is disrupted by cation binding. This dynamic process may be essential to facilitating the molecular packing and optical function of the lens.
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Cristalino , beta-Cristalinas , Cationes Bivalentes , Calcio , Sales (Química) , Calcio de la DietaRESUMEN
The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens, and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some disorders, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that compose these tissues, we generated an anterior segment cell atlas of the human eye using high-throughput single-nucleus RNA sequencing (snRNAseq). We profiled 195,248 nuclei from nondiseased anterior segment tissues of six human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in the lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment, revealing that some cell types are unique to a single structure, whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified through either Mendelian inheritance patterns or genome-wide association studies.
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Segmento Anterior del Ojo , Oftalmopatías , Adulto , Segmento Anterior del Ojo/citología , Segmento Anterior del Ojo/metabolismo , Humor Acuoso/citología , Humor Acuoso/metabolismo , Atlas como Asunto , Cuerpo Ciliar/citología , Cuerpo Ciliar/metabolismo , Oftalmopatías/genética , Estudio de Asociación del Genoma Completo , Humanos , Iris/citología , Especificidad de ÓrganosRESUMEN
The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm and is regulated by transcription factor Pax6 and secreted BMP4. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2, a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.
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Matriz Extracelular , Regulación del Desarrollo de la Expresión Génica , Cristalino , Factor de Transcripción PAX6 , Animales , Matriz Extracelular/metabolismo , Ratones , Cristalino/metabolismo , Cristalino/crecimiento & desarrollo , Cristalino/citología , Factor de Transcripción PAX6/metabolismo , Factor de Transcripción PAX6/genética , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/genética , Embrión de Pollo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción Paired Box/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Transducción de Señal , Pollos/genética , Ojo/metabolismo , Ojo/crecimiento & desarrollo , Ojo/embriologíaRESUMEN
Plasmonic nanoantennas have proven to be efficient transducers of electromagnetic to mechanical energy and vice versa. The sudden thermal expansion of these structures after an ultrafast optical pulsed excitation leads to the emission of hypersonic acoustic waves to the supporting substrate, which can be detected by another antenna that acts as a high-sensitivity mechanical probe due to the strong modulation of its optical response. Here, we propose and experimentally demonstrate a nanoscale acoustic lens comprised of 11 gold nanodisks whose collective oscillation at gigahertz frequencies gives rise to an interference pattern that results in a diffraction-limited surface acoustic beam of about 340 nm width, with an amplitude contrast of 60%. Via spatially decoupled pump-probe experiments, we were able to map the radiated acoustic energy in the proximity of the focal area, obtaining a very good agreement with the continuum elastic theory.
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A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < -0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.
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Epigénesis Genética , Cristalino , Multiómica , Regulación de la Expresión Génica , Diferenciación Celular/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cromatina/metabolismo , Cristalino/metabolismoRESUMEN
Connexin (Cx)-forming channels play essential roles in maintaining lens homeostasis and transparency. We showed here channel-independent roles of Cx50 in cell-cell adhesion and confirmed the second extracellular (E2) domain as a critical domain for cell adhesion function. We found that cell adhesion decreased in cells expressing chimeric Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with the Cx50 (E2) domain. This function is Cx channel-independent and Cx50 E2 domain-dependent cell adhesion acting in both homotypic and heterotypic manners. In addition, we generated eight site mutations of unique residues between Cx50 and the other two lens Cxs and found that mutation of any one of the residues abolished the adhesive function. Moreover, expression of adhesive-impaired mutants decreased adhesion-related proteins, N-cadherin and ß-catenin. Expression of the adhesion-impaired Cx50W188P mutant in embryonic chick lens caused enlarged extracellular spaces, distorted fiber organization, delayed nuclear condensation, and cortical cataracts. In summary, the results from both in vitro and in vivo studies demonstrate the importance of the adhesive function of Cx50 in the lens.
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Adhesión Celular , Conexinas , Cristalino , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Conexinas/metabolismo , Proteínas del Ojo/metabolismo , Uniones Comunicantes/metabolismo , Cristalino/metabolismo , Cadherinas/metabolismoRESUMEN
Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.
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Catarata , Cristalinas , Minerales , Animales , Ratones , Calcio/metabolismo , Catarata/genética , Catarata/fisiopatología , Conexinas/genética , Conexinas/metabolismo , Cristalinas/genética , Cristalinas/metabolismo , Cristalino/patología , Minerales/metabolismo , Microtomografía por Rayos X , Modelos Animales de EnfermedadRESUMEN
Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.
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Calcinosis , Catarata , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Glucosa , Hiperglucemia , Factor 1 Inducible por Hipoxia , Cristalino , Humanos , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/genética , Calcinosis/etiología , Calcinosis/metabolismo , Calcinosis/patología , Catarata/etiología , Catarata/metabolismo , Catarata/patología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Glucosa/metabolismo , Hiperglucemia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Cristalino/metabolismo , Cristalino/patología , Osteocalcina/metabolismo , Osteocalcina/genética , Transducción de Señal , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Previously we showed hyperosmotic solution caused TRPV1-dependent NKCC1 activation in the lens by a mechanism that involved ERK1/2 signaling. In various tissues, integrins and the cytoskeletal network play a role in responses to osmotic stress. Here, we examined the association between integrins and TRPV1-dependent activation of NKCC1 in mouse lens epithelium. Wild-type (WT) lenses exposed to the integrin agonist leukadherin-1 (LA-1) for 10 min displayed a ~33% increase in the bumetanide-sensitive rate of Rb uptake indicating NKCC activation. Paclitaxel, a microtubule stabilizing agent, abolished the Rb uptake response. In primary cultured lens epithelium LA-1 caused a robust ERK1/2 activation response that was almost fully suppressed by paclitaxel. The TRPV1 agonist capsaicin caused a similar ERK1/2 activation response. Consistent with an association between integrins and TRPV1, the TRPV1 antagonist A889425 prevented the Rb uptake response to LA-1 as did the ERK inhibitor U0126. LA-1 did not increase Rb uptake by lenses from TRPV1 knockout mice. In cells exposed to a hyperosmotic stimulus, both the ERK1/2 activation and Rb uptake responses were prevented by paclitaxel. Taken together, the findings suggest TRPV1 activation is associated with integrins and the tubulin cytoskeleton. This aligned with the observation that LA-1 elicited a robust cytoplasmic calcium rise in cells from WT lenses but failed to increase calcium in cells from TRPV1 knockout lenses. The results are consistent with the notion that integrin activation by LA-1, or a hyperosmotic stimulus, causes TRPV1 channel opening and the consequent downstream activation of the ERK1/2 and NKCC1 responses.
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BACKGROUND: Posterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFß) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO. METHODS: RNA sequencing (RNA-seq) was performed on both TGF-ß2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Our analysis revealed 1253 DEGs between TGF-ß2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq. CONCLUSIONS: Our RNA-Seq data identified 38 ferroptosis-related genes in TGF-ß2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-ß2 stimulation.
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Opacificación Capsular , Ferroptosis , Humanos , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/farmacología , Transcriptoma , Transición Epitelial-Mesenquimal/genética , Ferroptosis/genética , Western Blotting , Opacificación Capsular/genética , Opacificación Capsular/metabolismo , Células Epiteliales/metabolismoRESUMEN
Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.
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Acuaporinas , Conexinas , Proteínas del Ojo , Cristalino , Espectrometría de Masas , Acuaporinas/metabolismo , Acuaporinas/química , Proteínas del Ojo/metabolismo , Proteínas del Ojo/química , Animales , Espectrometría de Masas/métodos , Cristalino/metabolismo , Cristalino/química , Conexinas/metabolismo , Conexinas/química , Vimentina/metabolismo , Vimentina/química , Unión Proteica , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , alfa-Cristalinas/metabolismo , alfa-Cristalinas/químicaRESUMEN
Wearable soft contact lens sensors for continuous and nondestructive intraocular pressure (IOP) monitoring are highly desired as glaucoma and postoperative myopia patients grow, especially as the eyestrain crowd increases. Herein, a smart closed-loop system is presented that combines a Ti3C2Tx MXene-based soft contact lens (MX-CLS) sensor, wireless data transmission units, display, and warning components to realize continuous and nondestructive IOP monitoring/real-time display. The fabricated MX-CLS device exhibits an extremely high sensitivity of 7.483 mV mmHg-1, good linearity on silicone eyeballs, excellent stability under long-term pressure-release measurement, sufficient transparency with 67.8% transmittance under visible illumination, and superior biocompatibility with no discomfort when putting the MX-CLS sensor onto the Rabbit eyes. After integrating with the wireless module, users can realize real-time monitoring and warning of IOP via smartphones, the demonstrated MX-CLS device together with the IOP monitoring/display system opens up promising platforms for Ti3C2Tx materials as the base for multifunctional contact lens-based sensors and continuous and nondestructive IOP measurement system.
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Lentes de Contacto Hidrofílicos , Presión Intraocular , Titanio , Presión Intraocular/fisiología , Animales , Conejos , Titanio/química , Monitoreo Fisiológico/métodos , Monitoreo Fisiológico/instrumentaciónRESUMEN
The phase problem in the context of focusing synchrotron beams with X-ray lenses is addressed. The feasibility of retrieving the surface error of a lens system by using only the intensity of the propagated beam at several distances is demonstrated. A neural network, trained with a few thousand simulations using random errors, can predict accurately the lens error profile that accounts for all aberrations. It demonstrates the feasibility of routinely measuring the aberrations induced by an X-ray lens, or another optical system, using only a few intensity images.