Publications by authors named "Barry E Knox"

40 Publications

Retinal tissue preparation for high-resolution live imaging of photoreceptors expressing multiple transgenes.

MethodsX 2018 16;5:1140-1147. Epub 2018 Mar 16.

Departments of Neuroscience & Physiology, and Ophthalmology, SUNY Upstate Medical University, Syracuse, NY, United States.

Live imaging has become the favorite method in recent years to study the protein transport, localization and dynamics in live cells. Protein transport is extremely essential for proper function of photoreceptors. Aberration in the proper transport of proteins gives rise to the loss of photoreceptor and blindness. On the other hand, the ease of generation of transgenic tadpoles and the advantage of high resolution live confocal imaging provide new insight into understanding protein dynamics in photoreceptors. There are several steps for quantifying and visualizing fluorescently tagged proteins in photoreceptors starting with assembly of plasmids, generation of transgenic tadpoles, preparation of retinal tissues, imaging the transgenic photoreceptors and finally analyzing the recorded data. The focus of this manuscript is to describe how to prepare retinal tissues suited for live cell imaging and provide our readers with a tutorial video. We also give a summary of steps leading to a successful experiment that might be designed for imaging the ultrastructures of photoreceptors, the expression of two or more different fluorescently tagged proteins, their localization, distribution, or protein dynamics within photoreceptors. •Retinal tissue live imaging demonstrates the ultrastructures of photoreceptors.•High resolution live confocal imaging provides new insight into understanding the pathophysiology of photoreceptors.
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http://dx.doi.org/10.1016/j.mex.2018.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174271PMC
March 2018

Low-Temperature Trapping of Photointermediates of the Rhodopsin E181Q Mutant.

SOJ Biochem 2014;1(1)

Departments of Chemistry and Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.

Three active-site components in rhodopsin play a key role in the stability and function of the protein: 1) the counter-ion residues which stabilize the protonated Schiff base, 2) water molecules, and 3) the hydrogen-bonding network. The ionizable residue Glu-181, which is involved in an extended hydrogen-bonding network with Ser-186, Tyr-268, Tyr-192, and key water molecules within the active site of rhodopsin, has been shown to be involved in a complex counter-ion switch mechanism with Glu-113 during the photobleaching sequence of the protein. Herein, we examine the photobleaching sequence of the E181Q rhodopsin mutant by using cryogenic UV-visible spectroscopy to further elucidate the role of Glu-181 during photoactivation of the protein. We find that lower temperatures are required to trap the early photostationary states of the E181Q mutant compared to native rhodopsin. Additionally, a Blue Shifted Intermediate (BSI, λ = 498 nm, 100 K) is observed after the formation of E181Q Bathorhodopsin (Batho, λ = 556 nm, 10 K) but prior to formation of E181Q Lumirhodopsin (Lumi, λ = 506 nm, 220 K). A potential energy diagram of the observed photointermediates suggests the E181Q Batho intermediate has an enthalpy value 7.99 KJ/mol higher than E181Q BSI, whereas in rhodopsin, the BSI is 10.02 KJ/mol higher in enthalpy than Batho. Thus, the Batho to BSI transition is enthalpically driven in E181Q and entropically driven in native rhodopsin. We conclude that the substitution of Glu-181 with Gln-181 results in a significant perturbation of the hydrogen-bonding network within the active site of rhodopsin. In addition, the removal of a key electrostatic interaction between the chromophore and the protein destabilizes the protein in both the dark state and Batho intermediate conformations while having a stabilizing effect on the BSI conformation. The observed destabilization upon this substitution further supports that Glu-181 is negatively charged in the early intermediates of the photobleaching sequence of rhodopsin.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301618PMC
http://dx.doi.org/10.15226/2376-4589/1/1/00103DOI Listing
January 2014

Ablation of the proapoptotic genes CHOP or Ask1 does not prevent or delay loss of visual function in a P23H transgenic mouse model of retinitis pigmentosa.

PLoS One 2014 11;9(2):e83871. Epub 2014 Feb 11.

Departments of Neuroscience & Physiology, Biochemistry & Molecular Biology and Ophthalmology, Center for Vision Research, SUNY Upstate Medical University, Syracuse, New York, United States of America.

The P23H mutation in rhodopsin (Rho(P23H)) is a prevalent cause of autosomal dominant retinitis pigmentosa. We examined the role of the ER stress proteins, Chop and Ask1, in regulating the death of rod photoreceptors in a mouse line harboring the Rho(P23H) rhodopsin transgene (GHL(+)). We used knockout mice models to determine whether Chop and Ask1 regulate rod survival or retinal degeneration. Electrophysiological recordings showed similar retinal responses and sensitivities for GHL(+), GHL(+)/Chop(-/-) and GHL(+)/Ask1(-/-) animals between 4-28 weeks, by which time all three mouse lines exhibited severe loss of retinal function. Histologically, ablation of Chop and Ask1 did not rescue photoreceptor loss in young animals. However, in older mice, a regional protective effect was observed in the central retina of GHL(+)/Chop(-/-) and GHL(+)/Ask1(-/-), a region that was severely degenerated in GHL(+) mice. Our results show that in the presence of the Rho(P23H) transgene, the rate of decline in retinal sensitivity is similar in Chop or Ask1 ablated and wild-type retinas, suggesting that these proteins do not play a major role during the acute phase of photoreceptor loss in GHL(+) mice. Instead they may be involved in regulating secondary pathological responses such as inflammation that are upregulated during later stages of disease progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083871PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3921110PMC
October 2014

Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors.

BMC Mol Biol 2014 Feb 6;15. Epub 2014 Feb 6.

Departments of Neuroscience & Physiology, Ophthalmology and Biochemistry & Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.

Background: In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking.

Results: We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element.

Conclusions: We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.
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http://dx.doi.org/10.1186/1471-2199-15-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937059PMC
February 2014

An inducible expression system to measure rhodopsin transport in transgenic Xenopus rod outer segments.

PLoS One 2013 6;8(12):e82629. Epub 2013 Dec 6.

Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States of America.

We developed an inducible transgene expression system in Xenopus rod photoreceptors. Using a transgene containing mCherry fused to the carboxyl terminus of rhodopsin (Rho-mCherry), we characterized the displacement of rhodopsin (Rho) from the base to the tip of rod outer segment (OS) membranes. Quantitative confocal imaging of live rods showed very tight regulation of Rho-mCherry expression, with undetectable expression in the absence of dexamethasone (Dex) and an average of 16.5 µM of Rho-mCherry peak concentration after induction for several days (equivalent to >150-fold increase). Using repetitive inductions, we found the axial rate of disk displacement to be 1.0 µm/day for tadpoles at 20 °C in a 12 h dark /12 h light lighting cycle. The average distance to peak following Dex addition was 3.2 µm, which is equivalent to ~3 days. Rods treated for longer times showed more variable expression patterns, with most showing a reduction in Rho-mCherry concentration after 3 days. Using a simple model, we find that stochastic variation in transgene expression can account for the shape of the induction response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082629PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3857830PMC
October 2014

Regulation of rhodopsin-eGFP distribution in transgenic xenopus rod outer segments by light.

PLoS One 2013 15;8(11):e80059. Epub 2013 Nov 15.

Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology, and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States of America.

The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial variation of fluorescence intensity in cells expressing a fluorescently tagged rhodopsin transgene. There was a light synchronized fluctuation in intensity, with higher intensity in disks formed at night and lower intensity for those formed during the day. This fluctuation was absent in constant light or dark conditions. There was also a slow modulation of the overall expression level that was not synchronized with the lighting cycle or between cells in the same retina. The axial variations of other membrane-associated fluorescent proteins, eGFP-containing two geranylgeranyl acceptor sites and eGFP fused to the transmembrane domain of syntaxin, were greatly reduced or not detectable, respectively. In acutely light-adapted rods, an arrestin-eGFP fusion protein also exhibited axial variation. Both the light-sensitive Rho-eGFP and arrestin-eGFP banding were in phase with the previously characterized birefringence banding (Kaplan, Invest. Ophthalmol. Vis. Sci. 21, 395-402 1981). In contrast, endogenous rhodopsin did not exhibit such axial variation. Thus, there is an axial inhomogeneity in membrane composition or structure, detectable by the rhodopsin transgene density distribution and regulated by the light cycle, implying a light-regulated step for disk assembly in the OS. The impact of these results on the use of chimeric proteins with rhodopsin fused to fluorescent proteins at the carboxyl terminus is discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080059PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829889PMC
July 2014

Can fly photoreceptors lead to treatments for rho ((P23H)) -linked retinitis pigmentosa?

J Ophthalmic Vis Res 2013 Jan;8(1):86-91

SUNY Eye Institute, Department of Ophthalmology and Department of Neuroscience and Physiology, and Center for Vision Research SUNY Upstate Medical University, Syracuse, NY, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691985PMC
January 2013

A conserved aromatic residue regulating photosensitivity in short-wavelength sensitive cone visual pigments.

Biochemistry 2013 Jul 16;52(30):5084-91. Epub 2013 Jul 16.

Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology, and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210, United States.

Visual pigments have a conserved phenylalanine in transmembrane helix 5 located near the β-ionone ring of the retinal chromophore. Site-directed mutants of this residue (F207) in a short-wavelength sensitive visual pigment (VCOP) were studied using UV-visible spectroscopy to investigate its role in photosensitivity and formation of the light-activated state. The side chain is important for pigment formation: VCOP(F207A), VCOP(F207L), VCOP(F207M), and VCOP(F207W) substitutions all bound 11-cis-retinal and formed a stable visual pigment, while VCOP(F207V), VCOP(F207S), VCOP(F207T), and VCOP(F207Y) substitutions do not. The extinction coefficients of all pigments are close, ranging between 35800 and 45600 M⁻¹ cm⁻¹. Remarkably, the mutants exhibit an up to 5-fold reduction in photosensitivity and also abnormal photobleaching behavior. One mutant, VCOP(F207A), forms an isomeric composition of the retinal chromophore after illumination comparable to that of wild-type VCOP yet does not release the all-trans-retinal chromophore. These findings suggest that the conserved F207 residue is important for a normal photoactivation pathway, formation of the active conformation and the exit of all-trans-retinal from the chromophore-binding pocket.
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http://dx.doi.org/10.1021/bi400490gDOI Listing
July 2013

Modeling the flexural rigidity of rod photoreceptors.

Biophys J 2013 Jan;104(2):300-12

Departments of Neuroscience and Physiology, and Ophthalmology, State University of New York Upstate Medical University, Syracuse, New York, USA.

In vertebrate eyes, the rod photoreceptor has a modified cilium with an extended cylindrical structure specialized for phototransduction called the outer segment (OS). The OS has numerous stacked membrane disks and can bend or break when subjected to mechanical forces. The OS exhibits axial structural variation, with extended bands composed of a few hundred membrane disks whose thickness is diurnally modulated. Using high-resolution confocal microscopy, we have observed OS flexing and disruption in live transgenic Xenopus rods. Based on the experimental observations, we introduce a coarse-grained model of OS mechanical rigidity using elasticity theory, representing the axial OS banding explicitly via a spring-bead model. We calculate a bending stiffness of ∼10(5) nN⋅μm2, which is seven orders-of-magnitude larger than that of typical cilia and flagella. This bending stiffness has a quadratic relation to OS radius, so that thinner OS have lower fragility. Furthermore, we find that increasing the spatial frequency of axial OS banding decreases OS rigidity, reducing its fragility. Moreover, the model predicts a tendency for OS to break in bands with higher spring number density, analogous to the experimental observation that transgenic rods tended to break preferentially in bands of high fluorescence. We discuss how pathological alterations of disk membrane properties by mutant proteins may lead to increased OS rigidity and thus increased breakage, ultimately contributing to retinal degeneration.
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http://dx.doi.org/10.1016/j.bpj.2012.11.3835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552261PMC
January 2013

Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors.

J Gen Physiol 2012 Sep 13;140(3):249-66. Epub 2012 Aug 13.

Department of Ophthalmology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.

G protein-coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell-imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti-sapphire laser focused to the diffraction limit, which produced small sub-cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient D(lat) = 0.130 ± 0.012 µm(2)s(-1). Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.
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http://dx.doi.org/10.1085/jgp.201210818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434098PMC
September 2012

Research beyond Walls: State University of New York (SUNY) Eye Institute.

J Ophthalmic Vis Res 2012 Jan;7(1):94-6

SUNY Eye Institute, Departments of Ophthalmology and Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381116PMC
January 2012

Endoplasmic Reticulum Stress and Unfolded Protein Response Pathways: Potential for Treating Age-related Retinal Degeneration.

J Ophthalmic Vis Res 2012 Jan;7(1):45-59

SUNY Eye Institute, Departments of Neuroscience and Physiology and Ophthalmology, SUNY Upstate Medical University, Syracuse, NY, USA.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) and their aggregation impair normal cellular function and can be toxic, leading to cell death. Prolonged expression of misfolded proteins triggers ER stress, which initiates a cascade of reactions called the unfolded protein response (UPR). Protein misfolding is the basis for a variety of disorders known as ER storage or conformational diseases. There are an increasing number of eye disorders associated with misfolded proteins and pathologic ER responses, including retinitis pigmentosa (RP). Herein we review the basic cellular and molecular biology of UPR with focus on pathways that could be potential targets for treating retinal degenerative diseases.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381108PMC
January 2012

Translational science in ophthalmology.

Authors:
Barry E Knox

J Ophthalmic Vis Res 2012 Jan;7(1)

SUNY Eye Institute, Departments of Neuroscience and Physiology and Ophthalmology, SUNY Upstate Medical University, Syracuse, NY, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381096PMC
January 2012

Generation of transgenic Xenopus using restriction enzyme-mediated integration.

Methods Mol Biol 2012 ;884:17-39

Department of Neuroscience & Physiology, SUNY Upstate Medical University, Syracuse, NY, USA.

Transgenesis, the process of incorporating an exogenous gene (transgene) into an organism's genome, is a widely used tool to develop models of human diseases and to study the function and/or regulation of genes. Generating transgenic Xenopus is rapid and involves simple in vitro manipulations, taking advantage of the large size of the amphibian egg and external embryonic development. Restriction enzyme-mediated integration (REMI) has a number of advantages for transgenesis compared to other methods used to produce transgenic Xenopus, including relative efficiency, higher transgene expression levels, fewer genetic chimera in founder transgenic animals, and near-complete germ-line transgene transmission. This chapter explains the REMI method for generating transgenic Xenopus laevis tadpoles, including improvements developed to enable studies in the mature retina.
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http://dx.doi.org/10.1007/978-1-61779-848-1_2DOI Listing
October 2012

An S-opsin knock-in mouse (F81Y) reveals a role for the native ligand 11-cis-retinal in cone opsin biosynthesis.

J Neurosci 2012 Jun;32(23):8094-104

Center for Neuroscience, University of California, Davis, California 95618, USA.

In absence of their natural ligand, 11-cis-retinal, cone opsin G-protein-coupled receptors fail to traffic normally, a condition associated with photoreceptor degeneration and blindness. We created a mouse with a point mutation (F81Y) in cone S-opsin. As expected, cones with this knock-in mutation respond to light with maximal sensitivity red-shifted from 360 to 420 nm, consistent with an altered interaction between the apoprotein and ligand, 11-cis-retinal. However, cones expressing F81Y S-opsin showed an ∼3-fold reduced absolute sensitivity that was associated with a corresponding reduction in S-opsin protein expression. The reduced S-opsin expression did not arise from decreased S-opsin mRNA or cone degeneration, but rather from enhanced endoplasmic reticulum (ER)-associated degradation of the nascent protein. Exogenously increased 11-cis-retinal restored F81Y S-opsin protein expression to normal levels, suggesting that ligand binding in the ER facilitates proper folding. Immunohistochemistry and electron microscopy of normal retinas showed that Mueller cells, which synthesize a precursor of 11-cis-retinal, are closely adjoined to the cone ER, so they could deliver the ligand to the site of opsin synthesis. Together, these results suggest that the binding of 11-cis-retinal in the ER is important for normal folding during cone opsin biosynthesis.
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http://dx.doi.org/10.1523/JNEUROSCI.0131-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381355PMC
June 2012

Site-specific transgenesis in Xenopus.

Genesis 2012 Mar 15;50(3):325-32. Epub 2012 Feb 15.

Department of Ophthalmology, The Center for Vision Research and the SUNY Eye Institute, Upstate Medical University, Syracuse, NY 13210, USA.

Transgenesis is an essential, powerful tool for investigating gene function and the activities of enhancers, promoters, and transcription factors in the chromatin environment. In Xenopus, current methods generate germ-line transgenics by random insertion, often resulting in mosaicism, position-dependent variations in expression, and lab-to-lab differences in efficiency. We have developed and tested a Xenopus FLP-FRT recombinase-mediated transgenesis (X-FRMT) method. We demonstrate transgenesis of Xenopus laevis by FLP-catalyzed recombination of donor plasmid cassettes into F(1) tadpoles with host cassette transgenes. X-FRMT provides a new method for generating transgenic Xenopus. Once Xenopus lines harboring single host cassettes are generated, X-FRMT should allow for the targeting of transgenes to well-characterized integration site(s), requiring no more special reagents or training than that already common to most Xenopus labs.
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http://dx.doi.org/10.1002/dvg.22006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294184PMC
March 2012

Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes.

PLoS One 2012 19;7(1):e30101. Epub 2012 Jan 19.

Center for Vision Research, Department of Neuroscience, State University of New York Upstate Medical University, Syracuse, New York, United States of America.

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H)-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. Rho(P23H)-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H)-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H)-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H), suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030101PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261860PMC
June 2012

Rapid release of retinal from a cone visual pigment following photoactivation.

Biochemistry 2012 May 7;51(20):4117-25. Epub 2012 May 7.

Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.

As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated release of retinal from a short-wavelength-sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t(1/2)) of release of retinal from VCOP was 7.1 s, 250-fold faster than that of rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t(1/2) decreasing from 23 to 4 s with the pH decreasing from 4.1 to 8, respectively. However, the Arrhenius activation energy (E(a)) for VCOP derived from kinetic measurements between 4 and 20 °C was 17.4 kcal/mol, similar to the value of 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D(2)O) effect in VCOP, but this effect was smaller than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOP(D108A)) produced a pigment with an unprotonated chromophore (λ(max) = 360 nm) and dramatically slowed (t(1/2) ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D and D108A) was designed to move the counterion one α-helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (λ(max) = 420 nm). Moreover, the VCOP(S85D/D108A) mutant had retinal release kinetics (t(1/2) = 7 s) and an E(a) (18 kcal/mol) similar to those of the native pigment exhibiting no pH dependence. By contrast, the single mutant VCOP(S85D) had an ~3-fold decreased retinal release rate compared to that of the native pigment. Photoactivated VCOP(D108A) had kinetics comparable to those of a rhodopsin counterion mutant, Rho(E113Q), both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis but rather from differences in the properties of noncovalent binding of the retinal chromophore to the protein.
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http://dx.doi.org/10.1021/bi201522hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607377PMC
May 2012

Conserved residues in the extracellular loops of short-wavelength cone visual pigments.

Biochemistry 2011 Aug;50(32):6763-73

Department of Biochemistry and Molecular Biology and Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210, United States.

The role of the extracellular loop region of a short-wavelength sensitive pigment, Xenopus violet cone opsin, is investigated via computational modeling, mutagenesis, and spectroscopy. The computational models predict a complex H-bonding network that stabilizes and connects the EC2-EC3 loop and the N-terminus. Mutations that are predicted to disrupt the H-bonding network are shown to produce visual pigments that do not stably bind chromophore and exhibit properties of a misfolded protein. The potential role of a disulfide bond between two conserved Cys residues, Cys(105) in TM3 and Cys(182) in EC2, is necessary for proper folding and trafficking in VCOP. Lastly, certain residues in the EC2 loop are predicted to stabilize the formation of two antiparallel β-strands joined by a hairpin turn, which interact with the chromophore via H-bonding or van der Waals interactions. Mutations of conserved residues result in a decrease in the level of chromophore binding. These results demonstrate that the extracellular loops are crucial for the formation of this cone visual pigment. Moreover, there are significant differences in the structure and function of this region in VCOP compared to that in rhodopsin.
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http://dx.doi.org/10.1021/bi101557mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518856PMC
August 2011

Glutamic acid 181 is negatively charged in the bathorhodopsin photointermediate of visual rhodopsin.

J Am Chem Soc 2011 Mar 14;133(9):2808-11. Epub 2011 Feb 14.

Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269, USA.

Assignment of the protonation state of the residue Glu-181 is important to our understanding of the primary event, activation processes and wavelength selection in rhodopsin. Despite extensive study, there is no general agreement on the protonation state of this residue in the literature. Electronic assignment is complicated by the location of Glu-181 near the nodal point in the electrostatic charge shift that accompanies excitation of the chromophore into the low-lying, strongly allowed ππ* state. Thus, the charge on this residue is effectively hidden from electronic spectroscopy. This situation is resolved in bathorhodopsin, because photoisomerization of the chromophore places Glu-181 well within the region of negative charge shift following excitation. We demonstrate that Glu-181 is negatively charged in bathorhodopsin on the basis of the shift in the batho absorption maxima at 10 K [λ(max) band (native) = 544 ± 2 nm, λ(max) band (E181Q) = 556 ± 3 nm] and the decrease in the λ(max) band oscillator strength (0.069 ± 0.004) of E181Q relative to that of the native protein. Because the primary event in rhodopsin does not include a proton translocation or disruption of the hydrogen-bonding network within the binding pocket, we may conclude that the Glu-181 residue in rhodopsin is also charged.
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http://dx.doi.org/10.1021/ja1094183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3050519PMC
March 2011

Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.

J Biol Chem 2009 Nov 28;284(47):32662-9. Epub 2009 Sep 28.

Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA.

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone- and rod-specific PDE6 gamma-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.
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http://dx.doi.org/10.1074/jbc.M109.049916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781681PMC
November 2009

Light responses in rods of vitamin A-deprived Xenopus.

Invest Ophthalmol Vis Sci 2009 Sep 30;50(9):4477-86. Epub 2009 Apr 30.

Department of Ophthalmology, SUNY Upstate Medical University, Center for Vision Research, Syracuse, New York 13210, USA.

Purpose: Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration.

Methods: The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry.

Results: The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)).

Conclusions: Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.
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http://dx.doi.org/10.1167/iovs.08-3186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792892PMC
September 2009

Regulatory sequences in the 3' untranslated region of the human cGMP-phosphodiesterase beta-subunit gene.

Invest Ophthalmol Vis Sci 2009 Jun 14;50(6):2591-8. Epub 2009 Feb 14.

Jules Stein Eye Institute, University of California, Los Angeles, California 90095-7008, USA.

Purpose: Rod cGMP-phosphodiesterase, a key enzyme in visual transduction, is important for retinal integrity and function. Mutations in the gene encoding the phosphodiesterase beta-subunit (PDEbeta) cause retinal degeneration in animals and humans. Here the authors tested the hypothesis that elements in the 3' untranslated region (3' UTR) of the PDEbeta gene are involved in the regulation of PDEbeta expression.

Methods: Involvement of the 3' UTR of PDEbeta mRNA in the regulation of PDEbeta expression was assessed by Y-79 retinoblastoma cells or the heads of Xenopus laevis tadpoles with constructs containing the SV40 or PDEbeta promoter, the luciferase cDNA, and either the SV40 or the PDEbeta 3' UTR (or fragments of its sequence).

Results: Compared with the SV40 3' UTR (used as control), the entire PDEbeta 3' UTR decreased reporter gene expression in Y-79 retinoblastoma cells as well as in SY5Y neuroblastoma and 293 human embryonic kidney cell lines. However, the authors observed that two 100-nucleotide fragments from the PDEbeta 3' UTR increased while its noncanonical poly-adenylation signal abolished reporter gene expression in Y-79 retinoblastoma cells and in ex vivo experiments using Xenopus tadpole heads. In particular, an 11-nucleotide element (EURE) in one of the 100-nucleotide fragments was responsible for the upregulation of luciferase expression.

Conclusions: These studies indicate that the 3' UTR of the PDEbeta mRNA is involved in the complex regulation of this gene's expression in the retina. Moreover, the results show that the PDEbeta poly-A signal has a dominant inhibitory effect over two other regions in the 3' UTR that stimulate gene expression.
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http://dx.doi.org/10.1167/iovs.08-2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713258PMC
June 2009

The outer segment serves as a default destination for the trafficking of membrane proteins in photoreceptors.

J Cell Biol 2008 Nov;183(3):485-98

Albert Eye Research Institute, Duke University Medical Center, Durham, NC 27710, USA.

Photoreceptors are compartmentalized neurons in which all proteins responsible for evoking visual signals are confined to the outer segment. Yet, the mechanisms responsible for establishing and maintaining photoreceptor compartmentalization are poorly understood. Here we investigated the targeting of two related membrane proteins, R9AP and syntaxin 3, one residing within and the other excluded from the outer segment. Surprisingly, we have found that only syntaxin 3 has targeting information encoded in its sequence and its removal redirects this protein to the outer segment. Furthermore, proteins residing in the endoplasmic reticulum and mitochondria were similarly redirected to the outer segment after removing their targeting signals. This reveals a pattern where membrane proteins lacking specific targeting information are delivered to the outer segment, which is likely to reflect the enormous appetite of this organelle for new material necessitated by its constant renewal. This also implies that every protein residing outside the outer segment must have a means to avoid this "default" trafficking flow.
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http://dx.doi.org/10.1083/jcb.200806009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575789PMC
November 2008

Bioinformatic identification of novel putative photoreceptor specific cis-elements.

BMC Bioinformatics 2007 Oct 22;8:407. Epub 2007 Oct 22.

Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, USA.

Background: Cell specific gene expression is largely regulated by different combinations of transcription factors that bind cis-elements in the upstream promoter sequence. However, experimental detection of cis-elements is difficult, expensive, and time-consuming. This provides a motivation for developing bioinformatic methods to identify cis-elements that could prioritize future experimental studies. Here, we use motif discovery algorithms to predict transcription factor binding sites involved in regulating the differences between murine rod and cone photoreceptor populations.

Results: To identify highly conserved motifs enriched in promoters that drive expression in either rod or cone photoreceptors, we assembled a set of murine rod-specific, cone-specific, and non-photoreceptor background promoter sequences. These sets were used as input to a newly devised motif discovery algorithm called Iterative Alignment/Modular Motif Selection (IAMMS). Using IAMMS, we predicted 34 motifs that may contribute to rod-specific (19 motifs) or cone-specific (15 motifs) expression patterns. Of these, 16 rod- and 12 cone-specific motifs were found in clusters near the transcription start site. New findings include the observation that cone promoters tend to contain TATA boxes, while rod promoters tend to be TATA-less (exempting Rho and Cnga1). Additionally, we identify putative sites for IL-6 effectors (in rods) and RXR family members (in cones) that can explain experimental data showing changes to cell-fate by activating these signaling pathways during rod/cone development. Two of the predicted motifs (NRE and ROP2) have been confirmed experimentally to be involved in cell-specific expression patterns. We provide a full database of predictions as additional data that may contain further valuable information. IAMMS predictions are compared with existing motif discovery algorithms, DME and BioProspector. We find that over 60% of IAMMS predictions are confirmed by at least one other motif discovery algorithm.

Conclusion: We predict novel, putative cis-elements enriched in the promoter of rod-specific or cone-specific genes. These are candidate binding sites for transcription factors involved in maintaining functional differences between rod and cone photoreceptor populations.
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http://dx.doi.org/10.1186/1471-2105-8-407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225425PMC
October 2007

Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch.

Biochemistry 2007 May 18;46(18):5330-40. Epub 2007 Apr 18.

Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.

Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.
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http://dx.doi.org/10.1021/bi700138gDOI Listing
May 2007

Nr2e3 and Nrl can reprogram retinal precursors to the rod fate in Xenopus retina.

Dev Dyn 2007 Jul;236(7):1970-9

Department of Biochemistry & Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.

Transformation of undifferentiated progenitors into specific cell types is largely dependent on temporal and spatial expression of a complex network of transcription factors. Here, we examined whether neural retina leucine zipper (Nrl) and photoreceptor-specific nuclear receptor Nr2e3 transcription factors contribute to cell fate determination. We cloned the Xenopus Nr2e3 gene and showed that its temporal and spatial expression is similar to its mammalian ortholog. We tested its in vivo function by misexpressing these transcription factors in Xenopus eye primordia, demonstrating that either human Nr2e3 or Nrl directed photoreceptor precursors to become rods at the expense of cones. Furthermore, overexpression of Xenopus Nrl dramatically increased the number of lens fibers, whereas human Nrl did not, suggesting evolutionary divergence of function of the Nrl gene family. Misexpression of Nrl and Nr2e3 together were more effective than either transcription factor alone in directing precursors to the rod fate.
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http://dx.doi.org/10.1002/dvdy.21128DOI Listing
July 2007

Shedding light on cones.

J Gen Physiol 2006 Apr;127(4):355-8

Department of Biochemistry and Department of Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.

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http://dx.doi.org/10.1085/jgp.200609528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151508PMC
April 2006

Crystal structure of cone arrestin at 2.3A: evolution of receptor specificity.

J Mol Biol 2005 Dec 2;354(5):1069-80. Epub 2005 Nov 2.

Department of Neuroscience and Cell Biology, University of Texas Medical Branch, and Sealy Center for Molecular Science & Structural Biology, Galveston, TX 77555, USA.

Arrestins play a fundamental role in the regulation and signal transduction of G protein-coupled receptors. Here we describe the crystal structure of cone arrestin at 2.3A resolution. The overall structure of cone visual arrestin is similar to the crystal structures of rod visual and the non-visual arrestin-2, consisting of two domains, each containing ten beta-sheets. However, at the tertiary structure level, there are two major differences, in particular on the concave surfaces of the two domains implicated in receptor binding and in the loop between beta-strands I and II. Functional analysis shows that cone arrestin, in sharp contrast to its rod counterpart, bound cone pigments and non-visual receptors. Conversely, non-visual arrestin-2 bound cone pigments, suggesting that it may also regulate phototransduction and/or photopigment trafficking in cone photoreceptors. These findings indicate that cone arrestin displays structural and functional features intermediate between the specialized rod arrestin and the non-visual arrestins, which have broad receptor specificity. A unique functional feature of cone arrestin was the low affinity for its cognate receptor, resulting in an unusually rapid dissociation of the complex. Transient arrestin binding to the photopigment in cones may be responsible for the extremely rapid regeneration and reuse of the photopigment that is essential for cone function at high levels of illumination.
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http://dx.doi.org/10.1016/j.jmb.2005.10.023DOI Listing
December 2005

Developmental regulation of calcium-dependent feedback in Xenopus rods.

J Gen Physiol 2004 Nov;124(5):569-85

Center for Vision Research, Weiskotten Hall, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA.

The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from approximately 1.1 s(-1) to 3.7 s(-1) 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s(-1) to 4.0 s(-1) 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase-activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was approximately 0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.
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http://dx.doi.org/10.1085/jgp.200409162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234010PMC
November 2004