Edward N. Pugh - US grants

DESCRIPTION (provided by applicant): Cone photoreceptor cells initiate vision in daytime, operating in light intensities that saturate rods and render them useless to the visual system. The ability of cones to escape saturation relies in large part upon specializations of proteins, including cone opsins, within the cone phototransduction cascade that function in synergy to preserve inward cGMP-sensitive current in strong light. During their biosynthesis, cone opsins have a surprising need for the visual chromophore, 11-cis retinal, to achieve proper folding, and our preliminary data suggests that they also possess a compensating capacity for proteolytic degradation of misfolded proteins. The proposed work will lead to a comprehensive, molecularly based account of these specializations within cones, and could be used in future work to both extend the operating range of the much more numerous rods, and develop strategies that facilitate the generation of properly folded opsins or increase the capacity to degrade misfolded opsins.

Many vertebrates--including various fish, amphibians and birds, but not humans-- have been proven in behavioral experiments to be capable making orienting responses based upon the angle of polarization of linearly polarized light. Although the biophysical basis of a similar capability in invertebrates is well established, there is no accepted hypothesis that explains the vertebrate ability. We hypothesize that in vertebrates the ability is conferred by the manner in which light is trapped and propagates in a unique class of photoreceptors possessed by all the vertebrates with the ability: the double cone. We propose to test the hypothesis that the double cone, with its approximately elliptical inner segment cross section, is a polarization detector both by solving numerically Maxwell's equations for modal propagation in a dielectric waveguide model of the double cones of Lepomis cyanellus (green sunfish), and by measuring directly the light power throughput through double cones as a function of input polarization. We further hypothesize that the expected weak polarization modulation of the individual double cone is greatly enhanced by a class of "polarization-opponent" neurons in the inner retina which receive opposite signed inputs from double cones with their major elliptical axes arranged in orthogonal "tetradic" mosaics in sunfish and other species. We propose to locate and record from these hypothetical inner retinal neurons with voltage- sensitive dyes. Finally, we hypothesize that the role of this system of polarization-opponent neurons is to serve as a common mode rejection system for randomly polarized light (such as the underwater spacelight), and to confer on the animals which possess it polarization contrast sensitivity. This latter constitutes a heretofore undescribed kind of vision in vertebrates, and should enable those possessing it to segregate objects on the basis of the polarization distribution of the light reflected from them. We propose behavioral experiments in sunfish to characterize this predicted novel visual ability. Whilst the proposed work will have no immediate transfer to the study of human vision, we expect important spinoffs in the practical implementation of waveguide theory to human vision, in understanding underwater biology and ecology, and in instrumentation for stimulating and recording from tissue-mosaics.

DESCRIPTION: (Adapted from applicant's abstract) The ERG is the method of choice for rapid phenotyping of mice with mutations in retina-related genes. This is due to its ease of use, its inherently quantitative character, and its capacity to report alterations in many functionally important features of specific retinal cell types. These cells include: rods, the dominant cell type in the retina; cones, the basis of daytime-vision; and secondary neurons with which rods and cones communicate their respective signals. Building on previous work by the investigators, the proposed research will perfect rapid protocols for quantitative phenotyping of mice with ERG components attributable to these cell types. This will include production of light stimulation standards and standard values for each measurable ERG parameter for the most widely used wild type (WT) mouse strains. Knowledge of the pupillary response is essential for quantifying retinal stimulation in most conditions, and the pupillary response provides intrinsically quantitative input/output information about the integrity of a well understood visual system circuit. Illumination rearing conditions have profound effects on murine retinal health and function in electroretinography. Therefore, pupillometry and retinal histology will be used to quantify the effects of rearing illumination history and to chart the natural developmental course of the phenotyping parameters of WT mice from 2 weeks to 1 year of age. The research program will also develop a computer-controlled, unified ERG/pupillometry apparatus, which will incorporate all necessary features for mass-phenotyping. This proposal will also address data collection, storage, analysis, dissemination, and archiving.

The long-term goals of the University of Pennsylvania Nanomedicine Development Center (NDC) are to characterize quantitatively at the molecular scale the components and functional organization of selected supra-molecular cellular compartments (SMCCs), and to develop the nanoscale tools required to repair or replace these components in diseased cells. Many developmental and acquired diseases are caused by deficits in assembly or repair of specialized SMCCs. The molecular details of how cells accomplish the programmed self-assembly of complex compartments, such as photoreceptor outer segments, primary cilia in the renal tubules, the nerve growth cone, and the myofibril, have tremendous health-care relevance and intrinsic nanotechnological interest. We will combine state-of-the-art molecular biologic techniques with both existing and new nanotechniques to investigate the specific requirements of the individual SMCC components, the order of their assembly, their intramolecular contacts and interactions, and the crucial involvement of accessory proteins, such as chaperones and molecular motors. Improved understanding of these details will have two important outcomes: Firstly, replacement of defective components will be more readily targeted in disease processes, such as retinal degenerations, polycystic kidney disease and cardiomyopathy. Secondly, full understanding of the assembly requirements will allow design of practical, artificial nanotechnological devices, such as sensors, actuators and delivery vehicles, using the same principles of specificity and self-assembly. We will refine and develop several nanomedical tools for the proposed research. For example, we will perfect the two-photon confocal microscopy techniques required to quantitate individual SMCC components and follow their movements in real time within living cells. We will develop the use of synthetic polymersome nanoparticles to deliver fluorescently labeled SMCC proteins to living cells on the microscope stage for these experiments. We will use total internal reflection epifluorescence microscopy (TIRF) techniques to monitor cell-free assembly of SMCC component nanomachines. The use of modified polymersome and viral vector nanoparticles will be developed for delivery of essential SMCC components to cells in culture and in vivo. In addition, we will investigate new techniques for measurement of intracellular assembly of SMCC components at high resolution using nanotubes as optical sensors. These research tools will be useful for the study of multiple SMCCs, and broadly applicable to nanomedicine research in general.

[unreadable] DESCRIPTION (provided by applicant): Rod photoreceptors outnumber by 20:1 all other cell types in most vertebrate retinas. Arrestin is a 48 KDa protein expressed specifically in rod photoreceptor cells. Arrestin is the second-most abundant protein in the retina, estimated to be present at 1:10 to 1:2 the quantity of the most abundant retinal protein, rhodopsin, the visual pigment and G-protein coupled receptor that captures light and initiates the rod's light response. Arrestin plays an essential role in the inactivation of light-activated rhodopsin, capping the latter once it is phosphorylated. Arrestin was originally discovered and named retinal S-antigen, because it can evoke uveitis, an autoimmune inflammation of the blood-vessel rich layers of the eye. Arrestin undergoes a remarkable light-dependent migration between the two major compartments of the rod cell: in the dark-adapted eye it is concentrated largely in the inner segment region, while under light-adapted conditions it translocates almost completely to the outer segment, where rhodopsin resides. The proposed work investigates the molecular mechanisms underlying the light-dependent translocation of arrestin in living rod cells of the African clawed frog, Xenopus laevis, using arrestin-EGFP fusion proteins as tracers/biosensors, and 2-photon, confocal microscopy to quantify the protein and its movement. Work by several laboratories has shown such fusion proteins faithfully track the movement of native arrestin, and work from this laboratory has established that the dark-adapted distribution of arrestin is in disequilibrium with the cytoplasm. The proposed work will test several hypothesis about the nature of the arrestin dark disequilibrium and light-driven movement, including that (a) the disequilbrium is effected by a molecular "gate" in the connecting cilium; (b) the light-driven movement occurs purely by passive (diffusional) redistribution upon massive binding epitope creation by rhodopsin activation; (c) the redistribution is a light-activated process involving intraflagellar transport proteins, which serve to carry proteins from their origin to site of use in polarized, ciliated cells. [unreadable] [unreadable]

SUMMARY ? SMALL ANIMAL OCULAR IMAGING CORE The Small Animal Ocular Imaging Core (SAOIC) provides a wide and growing array of in vivo ocular imaging services to investigators who employ rodent models of ocular function and disease. The technological services include widefield Micron IVTM fundus camera video imaging (fluorescence and reflectance, with laser damage capability); cellular-level resolution Optical Coherence Tomography (OCT) and Scanning Laser Ophthalmoscopy (SLO); Adaptive Optics (AO) versions of both (AO-OCT, AO-SLO); combined OCT/SLO; and polarization- selective OCT. An especially noteworthy feature of the instrumentation is its ability to repeatedly image the same retinal locus ? and even the same cells ? in individual mice over months. The SAOIC Director and Managing Director provide experimental design consultation and tutorial sessions describing the capabilities of the instrumentation to potential investigators. Because the equipment is customized and relatively difficult to operate, and the datasets collected very large, the SAOIC staff also provide image analysis services when needed. Finally, the staff also trains the laboratory personnel of Core participating investigators to use key imaging tools (including the Micron IVTM camera, and the widefield OCT/SLO) when long-term studies are undertaken.