Exhumation and tectonic history of inaccessible subglacial interior East Antarctica from thermochronology on glacial erratics.

The geology, tectonic history and landscape evolution of ice-covered East Antarctica are the least known of any continent. Lithic boulders eroded from the continental interior and deposited in glacial moraines flanking the Transantarctic Mountains provide rare constraints on the geological history of central interior East Antarctica. Crystallization ages and ice velocities indicate these glacial erratics are not sourced locally from the Transantarctic Mountains but rather originate from the continental interior, possibly as far inland as the enigmatic Gamburtsev Subglacial Mountains. We apply low-temperature thermochronology to these boulders, including multi-kinetic inverse thermal modeling, to constrain a multi-stage episodic exhumation history. Cambro-Ordovician and Jurassic rapid-cooling episodes correlate with significant exhumation events accompanying Pan-African convergence and Gondwanan supercontinent rifting, respectively. Here we show that while Cretaceous rapid cooling overlaps temporally with Transantarctic Mountains formation, a lack of discrete younger rapid-cooling pulses precludes significant Cenozoic tectonic or glacial exhumation of central interior East Antarctica.

Aged Nrf2-Null Mice Develop All Major Types of Age-Related Cataracts.

Purpose: Age-related cataracts affect the majority of older adults and are a leading cause of blindness worldwide. Treatments that delay cataract onset or severity have the potential to delay cataract surgery, but require relevant animal models that recapitulate the major types of cataracts for their development. Unfortunately, few such models are available. Here, we report the lens phenotypes of aged mice lacking the critical antioxidant transcription factor Nfe2l2 (designated as Nrf2 -/-). Methods: Three independent cohorts of Nrf2 -/- and wild-type C57BL/6J mice were evaluated for cataracts using combinations of slit lamp imaging, photography of freshly dissected lenses, and histology. Mice were fed high glycemic diets, low glycemic diets, regular chow ad libitum, or regular chow with 30% caloric restriction. Results: Nrf2 -/- mice developed significant opacities between 11 and 15 months and developed advanced cortical, posterior subcapsular, anterior subcapsular, and nuclear cataracts. Cataracts occurred similarly in male mice fed high or low glycemic diets, and were also observed in 21-month male and female Nrf2 -/- mice fed ad libitum or 30% caloric restriction. Histological observation of 18-month cataractous lenses revealed significant disruption to fiber cell architecture and the retention of nuclei throughout the cortical region of the lens. However, fiber cell denucleation and initiation of lens differentiation was normal at birth, with the first abnormalities observed at 3 months. Conclusions: Nrf2 -/- mice offer a tool to understand how defective antioxidant signaling causes multiple forms of cataract and may be useful for screening drugs to prevent or delay cataractogenesis in susceptible adults.

Deletion of beaded filament proteins or the C-terminal end of Aquaporin 0 causes analogous abnormal distortion aberrations in mouse lens.

Lens-specific beaded filament (BF) proteins CP49 and filensin interact with the C-terminus of the water channel protein Aquaporin 0 (AQP0). Previously we have reported that a C-terminally end-deleted AQP0-expressing transgenic mouse model AQP0ΔC/ΔC developed abnormal optical aberrations in the lens. This investigation was undertaken to find out whether the total loss of the BF structural proteins alter the optical properties of the lens and cause optical aberrations similar to those in AQP0ΔC/ΔC lenses; also, to map the changes in the optical quality as a function of age in the single or double BF protein knockouts as well as to assess whether there is any significant change in the water channel function of AQP0 in these knockouts. A double knockout mouse (2xKO) model for CP49 and filensin was developed by crossing CP49-KO and filensin-KO mice. Wild type, CP49-KO, filensin-KO, and 2xKO lenses at different ages, and AQP0ΔC/ΔC lenses at postnatal day-17 were imaged through the optical axis and compared for optical quality and focusing property. All three knockout models showed loss of transparency, and development of abnormal optical distortion aberration similar to that in AQP0ΔC/ΔC. Copper grid focusing by the lenses at 6, 9 and 12 months of age showed an increase in aberrations as age advanced. With progression in age, the grid images produced by the lenses of all KO models showed a transition from a positive barrel distortion aberration to a pincushion distortion aberration with the formation of three distinct aberration zones similar to those produced by AQP0ΔC/ΔC lenses. Water permeability of fiber cell membrane vesicles prepared from CP49-KO, filensin-KO and 2xKO models, measured using the osmotic shrinking method, remained similar to that of the wild type without any statistically significant alteration (P > 0.05). Western blotting and quantification revealed the expression of comparable quantities of AQP0 in all three BF protein KOs. Our study reveals that loss of single or both beaded filament proteins significantly affect lens refractive index gradient, transparency and focusing ability in an age-dependent manner and the interaction of BF proteins with AQP0 is critical for the proper functioning of the lens. The presence of BF proteins is necessary to prevent abnormal optical aberrations and maintain homeostasis in the aging lens.

CP49 and filensin intermediate filaments are essential for formation of cold cataract.

Purpose: To investigate the molecular and cellular mechanisms of cataract induced by cold temperatures in young lenses of wild-type C57BL/6J (B6), wild-type 129SvJae (129), and filensin knockout (KO) mice. To determine how lens intermediate filament proteins, filensin (BFSP1) and CP49 (BFSP2), are involved in the formation of cold cataract. Methods: The formation of cold cataract was examined in enucleated lenses at different temperatures and was imaged under a dissecting microscope. Lens vibratome sections were prepared, immunostained with different antibodies and fluorescent probes, and then imaged with a laser confocal microscope to evaluate the protein distribution and the membrane and cytoskeleton structures in the lens fibers. Results: Postnatal day 14 (P14) wild-type B6 lenses showed cataracts dependent on cold temperatures in interior fibers about 420-875 µm (zone III) and 245-875 µm (zone II and zone III) from the lens surface, under 25 °C and 4 °C, respectively. In contrast, wild-type 129 (with CP49 gene deletion) and filensin KO (on the B6 background) lenses did not have cold cataracts at 25 °C but displayed a reduced cold cataract, especially in zone III, at 4 °C. Immunofluorescent staining data revealed that CP49 and filensin proteins were uniformly distributed in fiber cell cytosols without cold cataracts but accumulated or aggregated in the cell boundaries of the fibers where cold cataracts appeared. Conclusions: CP49 and filensin are important components for the formation of cold cataract in young B6 mouse lenses. Accumulated or aggregated CP49 and filensin beaded intermediate filaments in fiber cell boundaries might directly or indirectly contribute to the light scattering of cold cataract. Cold cataract in zone II is independent of beaded intermediate filaments. CP49 and filensin intermediate filaments and other lens proteins probably form distinct high molecular organizations to regulate lens transparency in interior fibers.

Completion of the Vimentin Rod Domain Structure Using Experimental Restraints: A New Tool for Exploring Intermediate Filament Assembly and Mutations.

Electron paramagnetic resonance (EPR) spectroscopy of full-length vimentin and X-ray crystallography of vimentin peptides has provided concordant structural data for nearly the entire central rod domain of the protein. In this report, we use a combination of EPR spectroscopy and molecular modeling to determine the structure and dynamics of the missing region and unite the separate elements into a single structure. Validation of the linker 1-2 (L1-2) modeling approach is demonstrated by the close correlation between EPR and X-ray data in the previously solved regions. Importantly, molecular dynamic (MD) simulation of the constructed model agrees with spin label motion as determined by EPR. Furthermore, MD simulation shows L1-2 heterogeneity, with a concerted switching of states among the dimer chains. These data provide the first ever experimentally driven model of a complete intermediate filament rod domain, providing research tools for further modeling and assembly studies.

Deletion of GLUT1 in mouse lens epithelium leads to cataract formation.

The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1loxP/loxP mice to generate Slc2a1loxP/loxP;GFAP-Cre+/0 (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.

Probing the Local Secondary Structure of Human Vimentin with Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Previously, an electron spin echo envelope modulation (ESEEM) spectroscopic approach was established to probe the local secondary structure of membrane proteins and peptides utilizing site-directed spin-labeling (SDSL). In this method, the side chain of one amino acid residue is selectively 2H-labeled and a nitroxide spin label is strategically placed 1, 2, 3, or 4 amino acids away from the 2H-labeled amino acid (denoted as i ± 1 to i ± 4, i represents the 2H-labeled amino acid). ESEEM can detect the dipolar coupling between the nitroxide spin label and 2H atoms on the amino acid side chain. Due to the periodicity of different secondary structures, different ESEEM patterns can be revealed to probe the structure. For an α-helical structural component, a 2H ESEEM signal can be detected for i ± 3 and i ± 4 samples, but not for i ± 1 or i ± 2 samples. Several 2H-labeled hydrophobic amino acids have been demonstrated in model system that can be utilized to identify local secondary structures via this ESEEM approach in an extremely efficient fashion. In this study, the ESEEM approach was used to investigate the rod 2B region of the full-length intermediate filament protein human vimentin. Consistent with previous EPR and X-ray crystallography results, our ESEEM results indicated helical structural components within this region. Thus, this ESEEM approach is able to identify α-helical structural components despite the coiled-coil nature of the vimentin structure. The data show that the human vimentin rod 2B adapted a typical α-helical structure around residue Leu309. This result is consistent with the X-ray data from fragmented protein segments and continuous wave EPR data on the full-length vimentin. Finally, the ESEEM data suggested that a local secondary structure slightly different from a typical α-helix was adopted around residue 340.

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

PURPOSE: To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein. METHODS: We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers. RESULTS: F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, ß2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, ß2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers. CONCLUSIONS: These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a ß2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.

Characterization of an Early-Onset, Autosomal Recessive, Progressive Retinal Degeneration in Bengal Cats.

PURPOSE: A form of retinal degeneration suspected to be hereditary was discovered in a family of Bengal cats. A breeding colony was established to characterize disease progression clinically, electrophysiologically, and morphologically, and to investigate the mode of inheritance. METHODS: Affected and related cats were donated by owners for breeding trials and pedigree analysis. Kittens from test and complementation breedings underwent ophthalmic and neuro-ophthalmic examinations and ERG, and globes were evaluated using light microscopy. RESULTS: Pedigree analysis, along with test and complementation breedings, indicated autosomal recessive inheritance and suggested that this disease is nonallelic to a retinal degeneration found in Persian cats. Mutation analysis confirmed the disease is not caused by CEP290 or CRX variants found predominantly in Abyssinian and Siamese cats. Ophthalmoscopic signs of retinal degeneration were noted at 9 weeks of age and became more noticeable over the next 4 months. Visual deficits were behaviorally evident by 1 year of age. Electroretinogram demonstrated reduced rod and cone function at 7 and 9 weeks of age, respectively. Rod responses were mostly extinguished at 14 weeks of age; cone responses were minimal by 26 weeks. Histologic degeneration was first observed at 8 weeks, evidenced by reduced photoreceptor numbers, then rapid deterioration of the photoreceptor layer and, subsequently, severe outer retinal degeneration. CONCLUSIONS: A recessively inherited primary photoreceptor degeneration was characterized in the Bengal cat. The disease is characterized by early onset, with histologic, ophthalmoscopic, and electrophysiological signs evident by 2 months of age, and rapid progression to blindness.

An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies.

PURPOSE: Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. METHODS: A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. RESULTS: An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence. CONCLUSIONS: A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

Role of Aquaporin 0 in lens biomechanics.

Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development.

SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes, as highlighted by the pleiotropic defects observed in Mowat-Wilson syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases, where it functionally links TGFß signaling to the loss of epithelial cell characteristics and gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers, such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation, and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes. Furthermore, 34% of the genes with increased expression in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes with decreased expression in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts.

Carbon turnover in the water-soluble protein of the adult human lens.

PURPOSE: Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the (14)C bomb pulse. METHODS: Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The (14)C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the (14)C bomb pulse. RESULTS: The water-insoluble fractions possessed (14)C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction. CONCLUSIONS: As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract.

Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly.

Very little data have been reported that describe the structure of the tail domain of any cytoplasmic intermediate filament (IF) protein. We report here the results of studies using site directed spin labeling and electron paramagnetic resonance (SDSL-EPR) to explore the structure and dynamics of the tail domain of human vimentin in tetramers (protofilaments) and filaments. The data demonstrate that in contrast to the vimentin head and rod domains, the tail domains are not closely apposed in protofilaments. However, upon assembly into intact IFs, several sites, including positions 445, 446, 451, and 452, the conserved "beta-site," become closely apposed, indicating dynamic changes in tail domain structure that accompany filament elongation. No evidence is seen for coiled-coil structure within the region studied, in either protofilaments or assembled filaments. EPR analysis also establishes that more than half of the tail domain is very flexible in both the assembly intermediate and the intact IF. However, by positioning the spin label at distinct sites, EPR is able to identify both the rod proximal region and sites flanking the beta-site motif as rigid locations within the tail. The rod proximal region is well assembled at the tetramer stage with only slight changes occurring during filament elongation. In contrast, at the beta site, the polypeptide backbone transitions from flexible in the assembly intermediate to much more rigid in the intact IF. These data support a model in which the distal tail domain structure undergoes significant conformational change during filament elongation and final assembly.

The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.

Despite the passage of ∼30 years since the complete primary sequence of the intermediate filament (IF) protein vimentin was reported, the structure remains unknown for both an individual protomer and the assembled filament. In this report, we present data describing the structure of vimentin linker 1 (L1) and rod 1B. Electron paramagnetic resonance spectra collected from samples bearing site-directed spin labels demonstrate that L1 is not a flexible segment between coiled-coils (CCs) but instead forms a rigid, tightly packed structure. An x-ray crystal structure of a construct containing L1 and rod 1B shows that it forms a tetramer comprising two equivalent parallel CC dimers that interact with one another in the form of a symmetrical anti-parallel dimer. Remarkably, the parallel CC dimers are themselves asymmetrical, which enables them to tetramerize rather than undergoing higher order oligomerization. This functionally vital asymmetry in the CC structure, encoded in the primary sequence of rod 1B, provides a striking example of evolutionary exploitation of the structural plasticity of proteins. EPR and crystallographic data consistently suggest that a very short region within L1 represents a minor local distortion in what is likely to be a continuous CC from the end of rod 1A through the entirety of rod 1B. The concordance of this structural model with previously published cross-linking and spectral data supports the conclusion that the crystallographic oligomer represents a native biological structure.

Long-term effects of intravitreal injection of GMP-grade bone-marrow-derived CD34+ cells in NOD-SCID mice with acute ischemia-reperfusion injury.

PURPOSE: To determine long-term safety of intravitreal administration of good manufacturing practice (GMP)-grade human bone-marrow-derived CD34(+) cells in NOD-SCID (nonobese diabetic-severe combined immunodeficiency) mice with acute retinal ischemia-reperfusion injury, a model for retinal vasculopathy. METHOD: Acute ischemia-reperfusion injury was induced in the right eye of adult NOD-SCID mice (n = 23) by transient elevation of intraocular pressure. Seven days later, 12 injured eyes and 5 normal contralateral eyes were injected each intravitreally with 5 × 10(4) CD34(+) cells isolated under GMP conditions from a healthy human donor bone marrow using an immunomagnetic cell isolation system. The remaining 11 injured eyes were not treated and served as controls. Mice were euthanized 1 day, 4 months, and 8 months later. Both eyes were enucleated and examined by immunohistochemical analysis and hematoxylin and eosin staining. Among mice followed for 8 months, electroretinography (ERG) was performed on both eyes before euthanization. All major organs were examined grossly and histologically after serial sectioning. RESULTS: Immunohistochemical staining 4 months after injection showed detectable CD34(+) cells in the retinal vasculature. ERG at 8 months after CD34(+) cell injection showed signals that were similar in untreated eyes. Histology of the enucleated eyes injected with CD34(+) cells showed no intraocular tumor or abnormal tissue growth after 8 months. Histologic analysis of all major organs showed no abnormal proliferation of human cells. CONCLUSIONS: Intravitreal administration of GMP-grade human bone-marrow-derived CD34(+) cells appears to be well tolerated long-term in eyes with acute retinal ischemic injury. A clinical trial will start to further explore this therapy.

Juvenile cataract-associated mutation of solute carrier SLC16A12 impairs trafficking of the protein to the plasma membrane.

PURPOSE: SLC16A12 encodes an orphan member of the monocarboxylate transporter family, MCT12. A nonsense mutation in SLC16A12 (c.643C>T; p.Q215X) causes juvenile cataract with a dominant inheritance pattern. In the present study, in vitro and in vivo experimental models were used to gain insight into how the SLC16A12 (c.643C>T) mutation leads to cataract formation. METHODS: MCT12 peptide antibodies were generated and used to examine the expression of MCT12 in the lens using immuno-confocal microscopy. To determine whether loss of Slc16a12 resulted in cataract formation, a Slc16a12 hypomorphic rat generated by transposon insertional mutagenesis was characterized using RT-PCR, slit lamp microscopy and histologic methods. Exogenous expression of MCT12 and MCT12:214Δ, a mimic of the mutant allele, were used to assess protein expression and trafficking. RESULTS: MCT12 protein was detected in the lens epithelium and secondary fiber cells at postnatal day 1. In the Slc16a12(TKO) rat, complete loss of MCT12 did not result in any detectable ocular phenotype. Exogenous expression of MCT12-GFP and MCT12:214Δ-GFP revealed that the full-length protein was trafficked to the plasma membrane (PM), whereas the truncated protein was retained in the endoplasmic reticulum (ER). When both MCT12 and MCT12:214Δ were coexpressed, to mimic the heterozygous patient genotype, the truncated protein was retained in the ER whereas full-length MCT12 was trafficked to the PM. Furthermore, MCT12 was identified as another MCT isoform that requires CD147 for trafficking to the cell surface. CONCLUSIONS: These data support a model whereby the SLC16A12 (c.643C>T) mutation causes juvenile cataract by a defect in protein trafficking rather than by haploinsufficiency.

Further analysis of the lens phenotype in Lim2-deficient mice.

PURPOSE: Lim2 (MP20) is the second most abundant integral protein of lens fiber cell membranes. A comparative analysis was performed of wild-type and Lim2-deficient (Lim2(Gt/Gt)) mouse lenses, to better define the anatomic and physiologic roles of Lim2. METHODS: Scanning electron microscopy (SEM) and confocal microscopy were used to assess the contribution of Lim2 to lens tissue architecture. Differentiation-dependent changes in cytoskeletal composition were identified by mass spectrometry and immunoblot analysis. The effects on cell-cell communication were quantified using impedance analysis. RESULTS: Lim2-null lenses were grossly normal. At the cellular level, however, subtle structural alterations were evident. Confocal microscopy and SEM analysis revealed that cortical Lim2(Gt/Gt) fiber cells lacked the undulating morphology that characterized wild-type fiber cells. On SDS-PAGE analysis the composition of cortical fiber cells from wild-type and Lim2-null lenses appeared similar. However, marked disparities were evident in samples prepared from the lens core of the two genotypes. Several cytoskeletal proteins that were abundant in wild-type core fiber cells were diminished in the cores of Lim2(Gt/Gt) lenses. Electrophysiological measurements indicated a small decrease in the membrane potential of Lim2(Gt/Gt) lenses and a two-fold increase in the effective intracellular resistivity. In the lens core, this may have reflected decreased expression levels of the gap junction protein connexin 46 (Cx46). In contrast, increased resistivity in the outer cell layers of Lim2(Gt/Gt) lenses could not be attributed to decreased connexin expression and may reflect the absence of cell fusions in Lim2(Gt/Gt) lenses. CONCLUSIONS: Comparative analysis of wild-type and Lim2-deficient lenses has implicated Lim2 in maintenance of cytoskeletal integrity, cell morphology, and intercellular communication.

Intermediate filaments regulate tissue size and stiffness in the murine lens.

PURPOSE: To define the contributions of the beaded filament (BF), a lens-specific intermediate filament (IF), to lens morphology and biomechanics. METHODS: Wild-type and congenic CP49 knockout (KO) mice were compared by using electrophysiological, biomechanical, and morphometric approaches, to determine changes that occurred because of the absence of this cytoskeletal structure. RESULTS: Electrophysiological assessment established that the fiber cells lacking the lens-specific IFs were indistinguishable from wild-type fiber cells. The CP49 KO mice exhibited lower stiffness, and an unexpected higher resilience than the wild-type lenses. The absence of these filaments resulted in lenses that were smaller, and exhibited a higher ratio of lens:lens nucleus size. Finally, lens shape differed as well, with the CP49 KO showing a higher ratio of axial:equatorial diameter. CONCLUSIONS: Previous work has shown that BFs are necessary in maintaining fiber cell and lens structural phenotypes with age, and that absence of these filaments results in a loss of lens clarity. This work demonstrates that several tissue-level properties that are critical to lens function are also dependent, at least in part, on the presence of these lens-specific IFs.

Site-directed spin labeling and electron paramagnetic resonance determination of vimentin head domain structure.

Intermediate filament (IF) proteins have been predicted to have a conserved tripartite domain structure consisting of a largely alpha-helical central rod domain, flanked by head and tail domains. However, crystal structures have not been reported for any IF or IF protein. Although progress has been made in determining central rod domain structure, no structural data have been reported for either the head or tail domains. We used site-directed spin labeling and electron paramagnetic resonance to analyze 45 different spin labeled mutants spanning the head domain of vimentin. The data, combined with results from a previous study, provide strong evidence that the polypeptide backbones of the head domains form a symmetric dimer of closely apposed backbones that fold back onto the rod domain, imparting an asymmetry to the dimer. By following the behavior of spin labels during the process of in vitro assembly, we show that head domain structure is dynamic, changing as a result of filament assembly. Finally, because the vimentin head domain is the major site of the phosphorylation that induces disassembly at mitosis, we studied the effects of phosphorylation on head domain structure and demonstrate that phosphorylation drives specific head domain regions apart. These data provide the first evidence-based model of IF head domain structure.