Biology subjects

Schey, K. L.

Publications and source records attributed to Schey, K. L..

5 recordsLinked to original sources

bioRxiv2021

Spatially-Resolved Proteomic Analysis of the Lens Extracellular Diffusion Barrier

PurposeThe presence of a physical barrier to molecular diffusion through lenticular extracellular space has been repeatedly detected in multiple species. This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and to direct nutrients into the lens core via the sutures at both poles. The purpose of this study is to characterize the molecular components that could contribute to the formation of this barrier. MethodsThree distinct regions in the bovine lens cortex were captured by laser capture microdissection guided by dye penetration. Proteins were digested by endoproteinase Lys C and trypsin. Mass spectrometry-based quantitative proteomic analysis followed by gene ontology (GO) and protein-protein interaction network analysis was performed. ResultsDye penetration showed that lens fiber cells first shrink the extracellular spaces of the broad sides of fiber cells followed by closure of the extracellular space between narrow sides at normalized lens distance (r/a) of 0.9. Accompanying the closure of extracellular space of the broad sides, dramatic proteomic changes were detected including up-regulation of several cell junctional proteins. AQP0 and its interacting partners ERM proteins were among a few proteins that were upregulated accompanying the closure of extracellular space of the narrow sides suggesting a particularly important role for the major lens membrane protein AQP0 in controlling the narrowing of the extracellular spaces between lens fiber cells. The results also provided important information related to biological processes that occur during fiber cell differentiation such as organelle degradation, cytoskeletal remodeling and GSH synthesis. ConclusionsThe formation of lens extracellular diffusion barrier is accompanied by significant membrane and cytoskeletal protein remodeling.

biochemistry↗

bioRxiv2021

Functional cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein

Hendra virus (HeV) and Nipah virus (NiV), the prototypic members of the Henipavirus (HNV) genus, are emerging, zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans (Eaton et al., 2006). While several research groups have made strides in developing candidate vaccines and therapeutics against henipaviruses, such countermeasures have not been licensed for human use, and significant gaps in knowledge about the human immune response to these viruses exist. To address these gaps, we isolated a large panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior occupation-related exposure to the equine HeV vaccine (Equivac(R) HeV). Competition-binding and hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies identified at least six distinct antigenic sites on the HeV/NiV receptor binding protein (RBP) that are recognized by human mAbs. Antibodies recognizing multiple antigenic sites potently neutralized NiV and/or HeV isolates in vitro. The most potent class of cross-reactive antibodies achieved neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3. Antibodies from this class mimic receptor binding by inducing a receptor-bound conformation to the HeV-RBP protein tetramer, exposing an epitope that appears to lie hidden in the interface between protomers within the HeV-RBP tetramer. Antibodies that recognize this cryptic epitope potently neutralized HeV and NiV. Flow cytometric studies using cell-surface-displayed HeV-RBP protein showed that cross-reactive, neutralizing mAbs from each of these classes cooperate for binding. In a highly stringent hamster model of NiVB infection, antibodies from both classes reduced morbidity and mortality and achieved synergistic protection in combination and provided therapeutic benefit when combined into two bispecific platforms. These studies identified multiple candidate mAbs that might be suitable for use in a cocktail therapeutic approach to achieve synergistic antiviral potency and reduce the risk of virus escape during treatment.

immunology↗

bioRxiv2020

Changes to zonular tension alters the subcellular distribution of AQP5 in regions of influx and efflux of water in the rat lens.

PurposeThe lens utilizes circulating fluxes of ions and water that enter the lens at both poles and exit at the equator to maintain its optical properties. We have mapped the subcellular distribution of the lens aquaporins (AQP0, 1, & 5) in these water influx and efflux zones and investigated how their membrane location is affected by changes in tension applied to the lens by the zonules. MethodsImmunohistochemistry using AQP antibodies was performed on axial sections obtained from rat lenses that had been removed from the eye and then fixed, or were fixed in situ to maintain zonular tension. Zonular tension was pharmacologically modulated by applying either tropicamide (increased), or pilocarpine (decreased). AQP labelling was visualized using confocal microscopy. ResultsModulation of zonular tension had no effect on AQP1 or AQP0 labelling in either the water efflux, or influx zones. In contrast, AQP5 labelling changed from membranous to cytoplasmic in response to both mechanical and pharmacologically induced reductions in zonular tension in both the efflux zone, and anterior (but not posterior) influx zone associated with the lens sutures. ConclusionsAltering zonular tension dynamically regulates the membrane trafficking of AQP5 in the efflux and anterior influx zones to potentially change the magnitude of circulating water fluxes in the lens.

physiology↗

bioRxiv2020

Localization of the Lens Intermediate Filament Switch by Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) enables targeted and untargeted visualization of the spatial localization of molecules in tissues with great specificity. The lens is a unique tissue that contains fiber cells corresponding to various stages of differentiation that are packed in a highly spatial order. The application of IMS to lens tissue localizes molecular features that are spatially related to the fiber cell organization. Such spatially resolved molecular information assists our understanding of lens structure and physiology; however, protein IMS studies are typically limited to abundant, soluble, low molecular weight proteins. In this study, a method was developed for imaging low solubility cytoskeletal proteins in the lens; a tissue that is filled with high concentrations of soluble crystallins. Optimized tissue washes combined with on-tissue enzymatic digestion allowed successful imaging of peptides corresponding to known lens cytoskeletal proteins. The resulting peptide signals facilitated segmentation of the bovine lens into molecularly distinct regions. A sharp intermediate filament transition from vimentin to lens-specific beaded filament proteins was detected in the lens cortex. MALDI IMS also revealed the region where posttranslational myristoylation of filensin occurs and the results indicate that truncation and myristoylation of filensin starts soon after filensin expression increased in the inner cortex. From intermediate filament switch to filensin truncation and myristoylation, multiple remarkable changes occur in the narrow region of lens cortex. MALDI images delineated the boundaries of distinct lens regions that will guide further proteomic and interactomic studies.

biochemistry↗

bioRxiv2020

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High Resolution Imaging Mass Spectrometry

The human retina evolved to facilitate complex visual tasks. It supports vision at light levels ranging from starlight to sunlight, and its supporting tissues and vasculature regulate plasma-delivered lipophilic essentials for vision, including retinoids (vitamin A derivatives). The human retina is of particular interest because of its unique anatomic specializations for high-acuity and color vision that are also vulnerable to prevalent blinding diseases. The retinas exquisite cellular architecture is composed of numerous cell types that are aligned horizontally, giving rise to structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. Suitable for retinal investigations, MALDI imaging mass spectrometry (IMS) technologies are now capable of providing images at low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach combined with a recently developed method of high accuracy multi-image registration was used to define the localization of lipids in human retina tissue at laminar, cellular, and sub-cellular levels. Data acquired by IMS combined with autofluorescence and bright-field microscopy of human retina sections in macular and peripheral regions indicate differences in distributions and abundances of lipid species across and within single cell types. Of note is localization of signals within specific layers of macula, localization within different compartments of photoreceptors and RPE, complementarity of signals between macular retina and non-macular RPE, and evidence that lipids differing by a single double bond can have markedly different distributions.

biochemistry↗