Autophagy Requirements for Eye Lens Differentiation and Transparency.

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation.

The role of FYCO1-dependent autophagy in lens fiber cell differentiation.

FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1-/-) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1-/- mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1-/- mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis.Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage.

Cardiac pacemaker cells (CPCs) rhythmically initiate the electrical impulses that drive heart contraction. CPCs display the highest rate of spontaneous depolarization in the heart despite being subjected to inhibitory electrochemical conditions that should theoretically suppress their activity. While several models have been proposed to explain this apparent paradox, the actual molecular mechanisms that allow CPCs to overcome electrogenic barriers to their function remain poorly understood. Here, we have traced CPC development at single-cell resolution and uncovered a series of cytoarchitectural patterning events that are critical for proper pacemaking. Specifically, our data reveal that CPCs dynamically modulate adherens junction (AJ) engagement to control characteristics including surface area, volume, and gap junctional coupling. This allows CPCs to adopt a structural configuration that supports their overall excitability. Thus, our data have identified a direct role for local cellular mechanics in patterning critical morphological features that are necessary for CPC electrical activity.

Novel mitochondrial derived Nuclear Excisosome degrades nuclei during differentiation of prosimian Galago (bush baby) monkey lenses.

The unique cellular organization and transparent function of the ocular lens depend on the continuous differentiation of immature epithelial cells on the lens anterior surface into mature elongated fiber cells within the lens core. A ubiquitous event during lens differentiation is the complete elimination of organelles required for mature lens fiber cell structure and transparency. Distinct pathways have been identified to mediate the elimination of non-nuclear organelles and nuclei. Recently, we reported the discovery of a unique structure in developing fiber cells of the chick embryo lens, called the Nuclear Excisosome, that is intractably associated with degrading nuclei during lens fiber cell differentiation. In the chick lens, the Nuclear Excisosome is derived from projections of adjacent cells contacting the nuclear envelope during nuclear elimination. Here, we demonstrate that, in contrast to the avian model, Nuclear Excisosomes in a primate model, Galago (bush baby) monkeys, are derived through the recruitment of mitochondria to form unique linear assemblies that define a novel primate Nuclear Excisosome. Four lenses from three monkeys aged 2-5 years were fixed in formalin, followed by paraformaldehyde, then processed for Airyscan confocal microscopy or transmission electron microscopy. For confocal imaging, fluorescent dyes labelled membranes, carbohydrate in the extracellular space, filamentous actin and nuclei. Fiber cells from Galago lenses typically displayed prominent linear structures within the cytoplasm with a distinctive cross-section of four membranes and lengths up to 30 µm. The outer membranes of these linear structures were observed to attach to the outer nuclear envelope membrane to initiate degradation near the organelle-free zone. The origin of these unique structures was mitochondria in the equatorial epithelium (not from plasma membranes of adjacent cells as in the chick embryo model). Early changes in mitochondria appeared to be the collapse of the cristae and modification of one side of the mitochondrial outer membrane to promote accumulation of protein in a dense cluster. As a mitochondrion surrounded the dense protein cluster, an outer mitochondrial membrane enclosed the protein to form a core and another outer mitochondrial membrane formed the outermost layer. The paired membranes of irregular texture between the inner core membrane and the outer limiting membrane appeared to be derived from modified mitochondrial cristae. Several mitochondria were involved in the formation and maturation of these unique complexes that apparently migrated around the fulcrum into the cytoplasm of nascent fiber cells where they were stabilized until the nuclear degradation was initiated. Thus, unlike in the chick embryo, the Galago lenses degraded nuclear envelopes with a Nuclear Excisosome derived from multiple mitochondria in the epithelium that formed novel linear assemblies in developing fiber cells. These findings suggest that recruitment of distinct structures is required for Nuclear Excisosome formation in different species.

The cause and consequence of fiber cell compaction in the vertebrate lens.

Fiber cells of the ocular lens are arranged in a series of concentric shells. New growth shells are added continuously to the lens surface and, as a consequence, the preexisting shells are buried. To focus light, the refractive index of the lens cytoplasm must exceed that of the surrounding aqueous and vitreous humors, and to that end, lens cells synthesize high concentrations of soluble proteins, the crystallins. To correct for spherical aberration, it is necessary that the crystallin concentration varies from shell-to-shell, such that cellular protein content is greatest in the center of the lens. The radial variation in protein content underlies the critical gradient index (GRIN) structure of the lens. Only the outermost shells of lens fibers contain the cellular machinery necessary for protein synthesis. It is likely, therefore, that the GRIN (which spans the synthetically inactive, organelle-free zone of the lens) does not result from increased levels of protein synthesis in the core of the lens but is instead generated through loss of volume by inner fiber cells. Because volume is lost primarily in the form of cell water, the residual proteins in the central lens fibers can be concentrated to levels of >500 mg/ml. In this short review, we describe the process of fiber cell compaction, its relationship to lens growth and GRIN formation, and offer some thoughts on the likely nature of the underlying mechanism.

Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells.

An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12-15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 µm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 µm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 µm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial-like projections appear to be initiated with a clathrin-like coat and driven by an internal actin network. In summary, a specialized cellular organelle, the nuclear excisosome, generated in part by adjacent fiber cells degrades nuclei during fiber cell differentiation and maturation.

Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus.

Defects in clearing apoptotic debris disrupt tissue and immunological homeostasis, leading to autoimmune and inflammatory diseases. Herein, we report that macrophages from lupus-prone MRL/lpr mice have impaired lysosomal maturation, resulting in heightened ROS production and attenuated lysosomal acidification. Impaired lysosomal maturation diminishes the ability of lysosomes to degrade apoptotic debris contained within IgG-immune complexes (IgG-ICs) and promotes recycling and the accumulation of nuclear self-antigens at the membrane 72 h after internalization. Diminished degradation of IgG-ICs prolongs the intracellular residency of nucleic acids, leading to the activation of Toll-like receptors. It also promotes phagosomal membrane permeabilization, allowing dsDNA and IgG to leak into the cytosol and activate AIM2 and TRIM21. Collectively, these events promote the accumulation of nuclear antigens and activate innate sensors that drive IFNα production and heightened cell death. These data identify a previously unidentified defect in lysosomal maturation that provides a mechanism for the chronic activation of intracellular innate sensors in systemic lupus erythematosus.

Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens.

PURPOSE: Increased use of phacoemulsification procedures for cataract surgeries has resulted in a dramatic decrease in the availability of cataractous nuclear specimens for basic research into the mechanism of human cataract formation. To overcome such difficulties, a fixation protocol was developed to provide good initial fixation of human donor lenses and extracted nuclei, when available, and is suitable for storing or shipping cataracts to laboratories where structural studies could be completed. METHODS: Cataractous lens nuclei (n=19, ages 12 to 74 years) were obtained from operating suites after extracapsular extraction. Transparent human donor lenses (n=27, ages 22 to 92 years) were obtained from the Ramayamma International Eye Bank. After the dimensions were measured with a digital caliper, samples were preserved in 10% formalin (neutral buffered) for 24 h and followed by fixation in 4% paraformaldehyde (pH 7.2) for 48 h. Samples were stored cold (4 °C) in buffer until shipped. Samples were photographed and measured before further processing for transmission electron microscopy. RESULTS: The dimensions of the samples varied slightly after short fixation followed by 1 to 5 months' storage before transmission electron microscopy processing. The mean change in the axial thickness of the donor lenses was 0.15±0.21 mm or 3.0±5.4%, while that of the extracted nuclei was 0.05±0.24 mm or 1.8±7.6%. Because the initial concern was whether the nuclear core was preserved, thin sections were examined from the embryonic and fetal nuclear regions. All cellular structures were preserved, including the cytoplasm, complex edge processes, membranes, and junctions. The preservation quality was excellent and nearly equivalent to preservation of fresh lenses even for the lens cortex. Cell damage characteristic of specific nuclear cataract types was easily recognized. CONCLUSIONS: The novel fixation protocol appears effective in preserving whole donor lenses and cataractous nuclei over a wide age range. Dimensions varied only 2%-3%, and fiber cell damage correlated well with standard fixation. These methods enable researchers and clinicians in remote settings to preserve donor lenses and rare examples of extracapsular extractions for detailed examination at later times.

Ultrastructural analysis of the human lens fiber cell remodeling zone and the initiation of cellular compaction.

The purpose is to determine the nature of the cellular rearrangements occurring through the remodeling zone (RZ) in human donor lenses, identified previously by confocal microscopy to be about 100 µm from the capsule. Human donor lenses were fixed with 10% formalin followed by 4% paraformaldehyde prior to processing for transmission electron microscopy. Of 27 fixed lenses, ages 22, 55 and 92 years were examined in detail. Overview electron micrographs confirmed the loss of cellular organization present in the outer cortex (80 µm thick) as the cells transitioned into the RZ. The transition occurred within a few cell layers and fiber cells in the RZ completely lost their classical hexagonal cross-sectional appearance. Cell interfaces became unusually interdigitated and irregular even though the radial cell columns were retained. Gap junctions appeared to be unaffected. After the RZ (40 µm thick), the cells were still irregular but more recognizable as fiber cells with typical interdigitations and the appearance of undulating membranes. Cell thickness was irregular after the RZ with some cells compacted, while others were not, up to the zone of full compaction in the adult nucleus. Similar dramatic cellular changes were observed within the RZ for each lens regardless of age. Because the cytoskeleton controls cell shape, dramatic cellular rearrangements that occur in the RZ most likely are due to alterations in the associations of crystallins to the lens-specific cytoskeletal beaded intermediate filaments. It is also likely that cytoskeletal attachments to membranes are altered to allow undulating membranes to develop.

Autophagy and mitophagy participate in ocular lens organelle degradation.

The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation.

Electron tomography of fiber cell cytoplasm and dense cores of multilamellar bodies from human age-related nuclear cataracts.

Human nuclear cataract formation is a multi-factorial disease with contributions to light scattering from many cellular sources that change their scattering properties over decades. The aging process produces aggregation of cytoplasmic crystallin proteins, which alters the protein packing and texture of the cytoplasm. Previous studies of the cytoplasmic texture quantified increases in density fluctuations in protein packing and theoretically predicted the corresponding scattering. Multilamellar bodies (MLBs) are large particles with a core of crystallin cytoplasm that have been suggested to be major sources of scattering in human nuclei. The core has been shown to condense over time such that the refractive index increases compared to the adjacent aged and textured cytoplasm. Electron tomography is used here to visualize the 3D arrangement of protein aggregates in aged and cataractous lens nuclear cytoplasm compared to the dense protein packing in the cores of MLBs. Thin sections, 70 nm thick, were prepared from epoxy-embedded human transparent donor lenses and nuclear cataracts. Tilt series were collected on an FEI T20 transmission electron microscope (TEM) operated at 200 kV using 15 nm gold particles as fiducial markers. Images were aligned and corrected with FEI software and reconstructed with IMOD and other software packages to produce animated tilt series and stereo anaglyphs. The 3D views of protein density showed the relatively uniform packing of proteins in aged transparent lens nuclear cytoplasm and less dense packing of aged cataractous cytoplasm where many low-density regions can be appreciated in the absence of the TEM projection artifacts. In contrast the cores of the MLBs showed a dense packing of protein with minimal density fluctuations. These observations support the conclusion that, during the nuclear cataract formation, alterations in protein packing are extensive and can result in pronounced density fluctuations. Aging causes the MLB cores to become increasingly different in their protein packing from the adjacent cytoplasm. These results support the hypothesis that the MLBs increase their scattering with age and nuclear cataract formation.

Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.

UNLABELLED: Current protocols for differentiation of stem cells make use of multiple treatments of soluble signals and/or matrix factors and result typically in partial differentiation to mature cells with under- or overexpression of adult tissue-specific genes. We developed a strategy for rapid and efficient differentiation of stem cells using substrata of biomatrix scaffolds, tissue-specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with a serum-free, hormonally defined medium (HDM) tailored for the adult cell type of interest. Biomatrix scaffolds were prepared by a novel, four-step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffolds maintained native histology, patent vasculatures, and ≈1% of the tissue's proteins but >95% of its collagens, most of the tissue's collagen-associated matrix components, and physiological levels of matrix-bound growth factors and cytokines. Collagens increased from almost undetectable levels to >15% of the scaffold's proteins with the remainder including laminins, fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix-bound cytokines and growth factors in patterns that correlate with histology. Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffolds and in an HDM tailored for adult liver cells, lost stem cell markers and differentiated to mature, functional parenchymal cells in ≈1 week, remaining viable and with stable mature cell phenotypes for more than 8 weeks. CONCLUSION: Biomatrix scaffolds can be used for biological and pharmaceutical studies of lineage-restricted stem cells, for maintenance of mature cells, and, in the future, for implantable, vascularized engineered tissues or organs.

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts.

The goal of this project was to determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 µm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming different RIs for the MLB and cytoplasm. In this study quantitative values of relative RI were determined from specific MLBs in electron micrographs of thin sections and used to calculate new Mie scattering plots. Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with osmium tetroxide and uranyl acetate, or uranyl acetate alone, prior to dehydration and embedding in epoxy or acrylic resins. Thin sections 70 nm thick were cut on a diamond knife and imaged without grid stains at 60 kV using a CCD camera on a transmission electron microscope (TEM). Integrated intensities in digital electron micrographs were related directly to protein density, which is linearly related to RI for a given substance. The RI of the MLB interior was calculated assuming an RI value of 1.42 for the cytoplasm from the literature. Calculated RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have interior protein densities similar to or less than the adjacent cytoplasm whereas others had significantly higher densities. The higher density MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. The bilayer coats were more often observed in MLBs from transparent donors and early stage cataracts indicating that bilayer loss was part of the MLB maturation, producing large low-density spaces around dense MLB cores. These spaces were frequently observed in advanced cataracts from India as large low-density crescents and annular rings. Predicted scattering from Mie plots using particles with dense cores and low-density rims was higher than reported previously, although the most important factor was the relative RI, not the MLB coat or lack thereof. In conclusion, the measurements confirm the high protein density and RI of some MLB interiors compared to adjacent cytoplasm. This high RI ratio used in the Mie calculations suggests that for 2000 MLBs/mm³, about half that reported for early stage nuclear cataracts from the US, the forward scattering could be more than 30% of the incident light. Therefore, the extent of forward scattering and its influence on macular visual acuity could be important components of ophthalmological evaluations of cataract patients.

Mie light scattering calculations for an Indian age-related nuclear cataract with a high density of multilamellar bodies.

PURPOSE: Multilamellar bodies (MLBs) are lipid-coated spheres (1-4 microm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses. Mie light scattering calculations have demonstrated that MLBs are potential sources of forward light scattering in human age-related nuclear cataracts due to their shape, size, frequency, and cytoplasmic contents, which often differ in refractive index from their surroundings. Previous studies have used data from several non-serial tissue sections viewed by light microscopy to extrapolate a volume and have assumed that MLBs are random in distribution. Currently, confocal microscopy is being used to examine actual tissue volumes from age-related nuclear cataracts and transparent lenses collected in India to confirm MLB shape, size, frequency, and randomness. These data allow Mie scattering calculations to be done with directly observed MLBs in intact tissue. METHODS: Whole Indian donor lenses and Indian lens nuclei after extracapsular cataract extraction were immersion-fixed in 10% formalin for 24 h and in 4% paraformaldehyde for 24 h before sectioning with a Vibratome. The 160 microm thick sections were stained for 24 h in the lipid dye DiI (1,1'-dilinoleyl-3,3,3',3' tetramethylindocarbocyanine, 4-chlorobenzenesulfonate), washed, stabilized in Permount under coverslips and examined with a Zeiss LSM 510 confocal microscope. Individual volumes of tissue (each typically 500,000 microm(3)) were examined using a plan-apochromat 63X oil (NA=1.4) lens. Other lenses were prepared for electron microscopy and histological examination using previously described procedures. RESULTS: Analysis of tissue volumes within Indian age-related nuclear cataracts and transparent lenses has confirmed that most MLBs are 1-4 microm in diameter and typically spherical with some occurring as doublets or in clusters. Most Indian cataracts and transparent lenses are similar to samples obtained in the United States. One cataract contained as many as 400,000 MLBs per mm(3) -100 times more than in cataracts collected in the United States. Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random. Mie calculations indicate that more than 90% of the incident light could be scattered by the high density of MLBs. CONCLUSIONS: An important finding was that one advanced Indian cataract contained many more MLBs than cataracts examined from India and previously from the United States. This indicates that specific conditions or susceptibilities may exist that promote the formation of excessive MLBs. Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity.

Palladin is an actin cross-linking protein that uses immunoglobulin-like domains to bind filamentous actin.

Palladin is a recently described phosphoprotein that plays an important role in cell adhesion and motility. Previous studies have shown that palladin overexpression results in profound changes in actin organization in cultured cells. Palladin binds to the actin-associated proteins alpha-actinin, vasodilator-stimulated phosphoprotein, profilin, Eps8, and ezrin, suggesting that it may affect actin organization indirectly. To determine its molecular function in generating actin arrays, we purified palladin and asked if it is also capable of binding to F-actin directly. In co-sedimentation and differential sedimentation assays, palladin was found to both bind and cross-link actin filaments. This bundling activity was confirmed by fluorescence and electron microscopy. Palladin fragments were then purified and used to determine the sequences necessary to bind and bundle F-actin. The Ig3 domain of palladin bound to F-actin, and a palladin fragment containing Ig3, Ig4, and the region linking these domains was identified as a fragment that was able to bundle F-actin. Because palladin has multiple Ig domains, and only one of them binds to F-actin, this suggests that different Ig domains may be specialized for distinct biological functions. In addition, our results suggest a potential role for palladin in generating specialized, actin-based cell morphologies via both direct actin cross-linking activity and indirect scaffolding activity.

Predicted light scattering from particles observed in human age-related nuclear cataracts using mie scattering theory.

PURPOSE: To employ Mie scattering theory to predict the light-scattering from micrometer-sized particles surrounded by lipid shells, called multilamellar bodies (MLBs), reported in human age-related nuclear cataracts. METHODS: Mie scattering theory is applicable to randomly distributed spherical and globular particles separated by distances much greater than the wavelength of incident light. With an assumed refractive index of 1.40 for nuclear cytoplasm, particle refractive indices from 1.33 to 1.58 were used to calculate scattering efficiencies for particle radii 0.05 to 3 microm and incident light with wavelengths (in vacuo) of 400, 550, and 700 nm. RESULTS: Surface plots of scattering efficiency versus particle radius and refractive index were calculated for coated spherical particles. Pronounced peaks and valleys identified combinations of particle parameters that produce high and low scattering efficiencies. Small particles (<0.3 microm radius) had low scattering efficiency over a wide range of particle refractive indices. Particles with radii 0.6 to 3 microm and refractive indices 0.08 to 0.10 greater (or less) than the surrounding cytoplasm had very high scattering efficiencies. This size range corresponds well to MLBs in cataractous nuclei (average MLB radius, 1.4 microm) and, at an estimated 4000 particles/mm(3) of tissue, up to 18% of the incident light was scattered primarily within a 20 degrees forward cone. CONCLUSIONS: The calculated size of spherical particles that scatter efficiently was close to the observed dimensions of MLBs in cataractous nuclei. Particle refractive indices only 0.02 units different from the surrounding cytoplasm scatter a significant amount of light. These results suggest that the MLBs observed in human age-related nuclear cataracts may be major sources of forward light scattering that reduces contrast of fine details, particularly under dim light.

Cryo-electron microscopy of biological samples.

A brief summary of current cryo-electron microscopy methods for processing and imaging biological tissues is provided. The main emphasis is given to two preparation procedures: frozen-hydrated samples because of the remarkable success of cryo-electron crystallography in obtaining near atomic resolution of integral membrane proteins, and high-pressure freezing because of the wide applicability for vitrification of large samples of normal and diseased tissues for ultrastructural and immunolabeling analysis. Methods for examining certain samples with a TEM cryo-stage are summarized. This includes an introduction to the relatively new area of cryo-electron tomography, which offers the possibility to observe the three-dimensional structure of subcellular components using only their natural variations in composition to generate contrast.

Cataract formation in a strain of rats selected for high oxidative stress.

The primary purpose of this study was to define the clinical and morphological features of cataractogenesis in the OXYS strain of rats that generate excess reactive oxygen species. Rats were sequentially examined from birth to the development of mature cataracts with slit lamp biomicroscopy. Morphology of selected stages of cataract development was studied using light and transmission electron microscopy (TEM), immunohistochemical localization of the lipid peroxidation product 4-hydroxynonenal (HNE) and fluorescent antibody labeling for DNA oxidation products. Lenses from age-matched normal rats were used as controls. OXYS rats developed cataracts as young as two weeks of age with progression to maturity by 1 year. Clinically, cataracts appeared initially either as nuclear or sub-capsular cortical changes and progressed to pronounced nuclear cataracts within months. TEM confirmed the light microscopic impression of region-specific alterations in both fiber cell cytoplasmic protein matrix and membrane structure. The outer adult nuclear region showed extensive cellular damage similar to osmotic cataracts, which is consistent with the postulated high uptake of glucose in the OXYS strain. The adult and outer fetal nuclear cells displayed several types of focal damage. The inner fetal and embryonic nuclear cells demonstrated textured cytoplasm, suggesting protein degradation or redistribution. Staining for HNE was increased in epithelium, cortex and nucleus compared to control lenses. Fluorescent antibody probes demonstrated increased levels of DNA oxidation products in OXYS rat lenses compared to age-matched controls. Fourier analysis of nuclear cytoplasm revealed significant components with corresponding sizes greater than 100 nm and, using a new theoretical approach, the texturing of the cytoplasm was shown to be sufficient to cause opacification of the nucleus. The OXYS rat appears to be an ideal model for oxidative stress cataractogenesis. The potential oxidative damage observed is extensive and characteristic of the developmental region. The source of oxidative damage may in part be a response to elevated levels of glucose. Because oxidative stress is thought to be a major factor in cataract formation in both diabetic and non-diabetic aging humans, this animal model may be a useful tool in assessing efficacy of antioxidant treatments that may slow or prevent cataract formation.

Distribution, spherical structure and predicted Mie scattering of multilamellar bodies in human age-related nuclear cataracts.

PURPOSE: To characterize multilamellar bodies (MLBs), determine their distribution along the optic axis and predict their potential Mie scattering within human age-related nuclear cataracts. Previous studies restricted to the equatorial plane have shown that MLBs are rare spherical objects that are 1-4 microm in diameter and covered by multiple layers of thin lipid-rich membranes. METHODS: Eight human aged transparent lenses were obtained from eye bank donors and eight human age-related nuclear cataracts were obtained immediately after extracapsular extraction. Each sample was Vibratome sectioned fresh into 200 microm thick sections that were fixed and embedded for light or electron microscopy. Light micrograph montages of the optic axis containing the juvenile, fetal and embryonic nuclei were examined. Mie scattering for random coated spherical particles was calculated based on assumed and measured particle parameters. RESULTS: Cells along the optic axis of the cataract contained approximately 7.5 times more MLBs as similar regions of the aged transparent lens, although these MLBs occurred with extremely low frequency. Cells of the aged transparent lens contained 1.3 MLBs mm(-2), while those of the cataract contained 9.6 MLBs mm(-2), which are equivalent to calculated densities of 5.6 x 10(2) and 4.1 x 10(3)mm(-3), respectively. While some MLBs were located within the cytoplasm near cell membranes, others were found away from membranes. The MLBs are distinct from circular profiles resulting from finger-like projections between adjacent cells. MLBs displayed varying geometries and cytoplasmic textures, although predominately spherical with interiors similar to adjacent fiber cell cytoplasm. These results are in agreement with previous theoretical analysis of light scattering from human lenses and with previous morphological studies examining the equatorial plane of the lens. Potential Mie scattering of spherical particles with the average properties of the observed MLBs and assumed refractive index properties was calculated to be forward scattering of as much as 20% of the incident light. CONCLUSIONS: The observed low frequency and absence of clustering of MLBs in the equatorial plane and along the optic axis suggests that MLBs are most likely uniformly distributed throughout the embryonic, fetal and juvenile nuclei of age-related cataracts. Because of their size, distribution, textured cytoplasm and calculated Mie scattering, MLBs probably cause local fluctuations in refractive index in human lens nuclei and, therefore, are potential sources of low-angle, forward light scattering that could impair image formation.

Ultrastructural characterization and Fourier analysis of fiber cell cytoplasm in the hyperbaric oxygen treated guinea pig lens opacification model.

The structural characteristics of differentiated fiber cells in control and hyperbaric oxygen (HBO)-treated guinea pig lenses were examined by transmission electron microscopy (TEM). Emphasis was placed on cell damage, membrane integrity, and cytoplasmic texture. Given the faint gross opacities observed in HBO-treated lenses in previous studies, it was hypothesized that subtle but significant morphological differences due to oxidative damage exist between control and treated animals. Experimental animals received either 70 or 85 treatments with HBO (2.5 atm of 100% O(2) for 2.5 hr, 3 times per week for 5-7 months). All specimens were obtained within 24 hr of death. Freshly cut Vibratome lens sections were fixed and processed for low and high-magnification thin-section TEM analysis. Cytoplasmic texture was analyzed using Fourier and autocorrelation image processing techniques. Low-magnification analysis revealed relatively insignificant differences in general appearance between the fiber cells of the inner fetal and embryonic nuclei in control and HBO-treated guinea pigs. Both groups demonstrated cells of similar morphology with equivalent membrane complexity and homogeneous cytoplasmic texture. Evidence of any major cellular damage or extracellular space debris was not obvious. High-magnification analysis of the cytoplasm of the treated lenses exhibited a mild, yet detectable increase in texture compared with controls and was confirmed by Fourier analysis. Cytoplasmic texture increased in complexity with additional treatments. The absence of major cellular damage in the lenses of HBO-treated animals suggests a less conspicuous source of light scattering. The small changes in cytoplasmic organization observed between treated and control animals may entirely account for the increase in nuclear light scattering observed by slit lamp. The results obtained with this guinea pig/HBO model parallel many of the morphological data associated with human nuclear cataracts. The high-angle scattering observed in the lens of the HBO-treated guinea pig may represent the type of cytoplasmic reorganization that occurs with mild oxidation, effectively making it a valuable model for human lens aging.