Visual Pigments and Light Detection in the Eye

Most forms of animal vision begin with light absorption by visual pigments in the eye. A typical visual pigment consists of a G protein-coupled receptor protein – opsin – covalently conjugated to a chromophore. Sub-families of opsins show distinctive physicochemical properties and cellular expressions, often attuned to the specific visual functions that they serve. Here, we examined a number of molecular and functional features of three sub-families of opsins. We found that: (1) an active molecule of rhodopsin (a ciliary opsin expressed in rod photoreceptors for dim-light vision) amplifies the light signal by activating about 20-30 transducin molecules at the peak of the current response to single photon-absorption. (2) the thermal activation of native and some mutant rhodopsin and cone pigments (ciliary opsins in cone photoreceptors for color vision) in the dark is indeed an isomerization event, the rate of which can be quantitatively predicted by multi-vibrational-mode statistical mechanics. (3) melanopsin, a rhabdomeric opsin that underlies the intrinsic photosensitivity of a subgroup of retinal ganglion cells and is responsible for diverse non-image-forming visual functions in mammals, is also expressed in some thick, myelinated neuronal processes in the rat iris that possibly originate from the trigeminal ganglia. (4) neuropsin (OPN5), a previous orphan opsin, mediates the photoentrainment of the local circadian rhythm in the mammalian retina and cornea

Time-resolved infrared absorption spectroscopy applied to photoinduced reactions: how and why

Abstract: Time-resolved infrared (IR) spectroscopy is a widely used technique in the investigation of photoinduced reactions, given its capabilities of providing structural information about the presence of intermediates and the reaction mechanism. Despite the fact that it is used in several fields since the ‘80s, the communication between the different scientific communities (photochemists, photobiologists, etc.) has been to date quite limited. In some cases, this lack of communication happened—and still happens—even inside the same scientific community (for instance between specialists in ultrafast ps/fs IR and those in “fast” ns/µs/ms IR). Even more surprising is the difficulty of non-specialists to understand the potential of time-resolved IR spectroscopy, despite the fact that IR spectroscopy is normally taught to all chemistry and material science students, and to several biology and physics students. This tutorial review aims at helping to solve these issues, first by providing a comprehensive but reader-friendly overview of the different techniques, and second, by focusing on five “case studies” (from photobiology, gas-phase photocatalysis, photochemistry, semiconductors and metal-carbonyl complexes). We are confident that this approach can help the reader—whichever is its background—to understand the capabilities of time-resolved IR spectroscopy to study the mechanism of photoinduced reactions. Graphical Abstract: [Figure not available: see fulltext.

Locally Learning Biomedical Data Using Diffusion Frames

Diffusion geometry techniques are useful to classify patterns and visualize high-dimensional datasets. Building upon ideas from diffusion geometry, we outline our mathematical foundations for learning a function on high-dimension biomedical data in a local fashion from training data. Our approach is based on a localized summation kernel, and we verify the computational performance by means of exact approximation rates. After these theoretical results, we apply our scheme to learn early disease stages in standard and new biomedical datasets

FTIR difference and resonance raman spectroscopy of rhodopsins with applications to optogenetics

Thesis (Ph. D.)--Boston UniversityThe major aim of this thesis is to investigate the molecular basis for the function of several types of rhodopsins with special emphasis on their application to the new field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7 a-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a light driven proton pump similar to the extensively studied bacteriorhodopsin (BR); channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII), a light-sensing protein that modulates phototaxis used in archaebacteria; and squid rhodopsins (sRho), the major photopigment in squid vision and a model for human melanopsin, which controls circadian rythms. The primary techniques used in these studies were FTIR difference spectroscopy and resonance Raman spectroscopy. These techniques, in combination with site directed mutagenesis and other biochemical methodologies produced new knowledge regarding the structural changes of the retinal chromophore, the location and function of internal water molecules as well as specific amino acids and peptide backbone. Specialized techniques were developed that allowed rhodopsins to be studied in intact membrane environments and in some cases in vivo measurements were made on rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting proteins and membrane potential to be investigated. Evidence was found that the local environment of one or more internal water molecules in SRII is altered by interaction with its cognate transducer, Htrii, and is also affected by the local lipid environment. In the case of AR3, many of the broad IR continuum absorption changes below 3000 cm-1, assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR, were found to be very similar to BR. Bands assigned to water molecules near the Schiff base postulated to be involved in proton transport were, however, shifted or absent. Structural changes of internal water molecules and possible bands associated with the interaction with ,8-arrestins were also detected in photoactivated squid rhodopsin when transformed to the acid Meta intermediate. Near-IR confocal resonance Raman measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. On the basis of these measurements, a model is proposed which provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential

Retinal function of the voltage-gated calcium channel subunit α2δ-3 / Light-dependent effects in α2δ-3 mutant and in wild type retina

The retina employs a large number of cell types that fulfill a broad spectrum of computations. It comes as no surprise that this complex network would make use of an equal diversity of molecular tools, such as voltage-gated calcium channels (VGCC). In fact, all pore-forming α1 subunits of VGCC and modulatory β and auxiliary α2δ subunits were found in the retina. Yet, little detail is known about the functional implementation of individual VGCC subunits in the retinal circuitry. My work described in part 1 focused on the retinal expression and function of one VGCC subunit, called α2δ-3, employing an α2δ-3 knockout mouse. I found transcription of all α2δ subunit genes throughout postnatal retinal development and strong expression of α2δ-3 in horizontal cells. Yet, in my patch-clamp recordings from isolated horizontal cells I did not find an impact on their somatic calcium currents, leaving a possible involvement of α2δ-3 in the horizontal cell axon-to-rod connection. Outer retina function, determined by electroretinogram, and optokinetic reflex behavior was normal in α2δ-3 knockout animals. However, I discovered changes to the retinal output in micro-electrode array recordings of ganglion cell responses. I applied a paradigm of light stimulation at different ambient luminance levels that revealed effects of the α2δ-3 knockout only in scotopic and mesopic light levels. In summary, α2δ-3 is a candidate for horizontal cell axon-specific calcium signal modulation and exerts its function in non-photopic regimes. The retina constantly adapts to features of the current visual environment, most prominently, the ambient light intensity or luminance. These adaptations are based on mechanisms throughout the retinal network. Adaption is commonly considered to keep signal processing within the dynamic range of the system as well as keep the retinal output stable across changing conditions, such as the light intensity. The results of part 1 show that different building blocks of retinal circuits - here the α2δ-3 subunit - can contribute to retinal function at distinct light level regimes. In part 2, we looked more generally at the output of the retina (responses of ganglion cells) across different levels of ambient luminance. We found that ganglion cell responses were not stable across luminance levels, neither in single ganglion cell types nor in the ganglion cell population, but that they changed their responses qualitatively. These response changes were also reflected downstream in the activity of the lateral geniculate nucleus. Furthermore, we observed that rod photoreceptors could drive visual responses of ganglion cells in photopic luminance levels, where they were commonly thought to be saturated. While experiencing initial incremental saturation upon stepping to photopic luminance, rods recovered responsiveness at all light levels tested, but the rate of recovery was faster with brighter ambient light intensity. Computational modeling suggested adaptive translocation of elements of the signal transduction cascade as potential explanations for rod signaling at high light intensities. The photopic rod activity dynamics have important implications for the interpretation of experimental data and for the question of rod photoreceptor contributions to daylight vision. In summary, while some circuitry elements associated with luminance regimes are known (e.g. rod and cone pathways), details on the underlying molecular mechanisms are scarce. My data suggests α2δ-3 as a promising candidate for a molecular determinant of light adaptation that could exert its function within horizontal cells in an axonal compartment-specific way. It will be interesting to pinpoint the exact role of α2δ- 3 in retinal light adaptation and to determine what (sub-)cellular function this protein serves in horizontal cells

Recovery of Spatial Vision Following Intense Light Adaptation

This thesis presents a series of investigations that describe the recovery of spatial vision following a period of intense light adaptation. The first investigation in this thesis (Chapter 2) shows, for the first time, how threshold to sinusoidal gratings (0.6-14 c.p.d.), presented in the parafovea, varies during the course of dark adaptation (30 min). For all subjects (n=7) threshold to low spatial frequency gratings declined throughout the experiment, showing discrete cone and rod limbs. For subjects with normal colour vision, threshold to high spatial frequency gratings (14 c.p.d.) increased during the ‘rod’ phase of dark adaptation, a phenomenon known as suppressive rod-cone interaction (SRCI). This increase in threshold, was not observed in the 2 protanopes studied which suggests a distil site for this interaction. From a functional perspective, the most important quantity for the visual system to extract form the environment is contrast. Therefore, a technique has been developed for measuring contrast sensitivity (CS) during recovery from a period of intense light adaptation. Results were obtained for 2 subjects, for 5 spatial frequencies (0.6-19.9 c.p.d.) at 5 luminances (50-0.005 cdm^-2)(Chapter 3). These results provide a practical description of visual performance under such conditions. The effect of ‘bleach’ intensity and duration on CS recovery was investigated in Chapter 4. Equal energy ‘bleaches’ that differed in intensity (I) and duration (t) (i.e. I x t = k) produced different CS recovery functions. This suggests that CS recovery is only partly explained by the concentration of a photoproduct (the photochemical hypothesis). The effect of ‘bleach’ intensity and duration on CS recovery was investigated in Chapter 4. Equal energy ‘bleaches’ that differed in intensity (I) and duration (t) (i.e. I x t = k) produced different CS recovery functions. This suggests that CS recovery is only partly explained by the concentration of a photoproduct (the photochemical hypothesis). To obtain a fuller understanding of how visual performance changes during photostress testing, 50 subjects were tested using a modified version of the CS recovery technique. Contrast sensitivity recovery functions were modelled using a single exponential function. Analysis of the model variables showed that the time constant of CS recovery increases with age. The investigations presented in this thesis extend the current understanding of the recovery of spatial vision following a period of intense light adaptation

Doctor of Philosophy

dissertationHuman retinitis pigmentosa (RP) typically involves decades of progressive vision loss before some patients become blind, and prospective therapies target patients who have been blind for substantial time, even decades. Evaluations of molecular and cellular therapies have primarily employed short-lived mouse models lacking the scope of remodeling common in human RP. The Rho Tg P347L transgenic rabbit offers a unique opportunity to evaluate the primary degeneration event and subsequent progressive remodeling that ensues over a timespan that recapitulates the human disease phenotype. Retinas from a TgP347L rabbit model of human dominant RP and wild-type litter mates were harvested over an 8-year span and processed for transmission electron microscope connectomics, immunocytochemistry for a range of macromolecules, and computational molecular phenotyping for small molecules, including transport tracing with D-Asp. Early time points in the TgP347L rabbit recapitulate the established sequence of photoreceptor loss, retinal remodeling, and reprogramming, and also reveal progressive disruptions in Müller cell metabolism, where rather than observing a homogeneous glial population, chaotic metabolic signatures emerge. By 4 years, virtually all remnants of photoreceptors are gone and the neural retina manifests severe cell loss and near complete loss of glutamine synthetase, though glial glutamate transport persists. By 6 years, there is a global >90% neuronal loss. In some regions the retina is devoid of identifiable cells and replaced by unknown debris-like assemblies. Though the 6-year retina does have locations with recognizable neurons, all cell types are drastically reduced in number and some have altered metabolic phenotypes. These results are never seen in wt littermates, including rabbits which are 8 years old. Electron microscopic analysis using wide-field connectomics imaging of the 6-year TgP347L sample demonstrates some structurally normal synapses, indicating that survivor neurons in these regions are not quiescent despite the lack of sensory input for a substantial period of time. These results indicate that, although photoreceptor degeneration is the trigger, retinal remodeling ultimately gives way to neurodegeneration, which is a separate unrelenting disease process independent of the initial insult, closely resembling slow progressive CNS neurodegenerations. Indeed, both metabolic disruption and debris-related degeneration predicts the existence of a persistent neuropathy, and increases in ?-synuclein levels support a proteinopathy component. Remodeling and neurodegeneration progress until the retina is devoid of recognizable cells. There is no stable state into which the retina settles and no cell type is spared. This has profound implications for current therapeutics. There will likely be critical windows for implementation but, ultimately, suspension of neurodegenerative remodeling will be required for long-term success

Single-Molecule Measurements of Complex Molecular Interactions in Membrane Proteins using Atomic Force Microscopy

Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage