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Featured researches published by James M. Morrow.


Visual Neuroscience | 2011

A novel rhodopsin-like gene expressed in zebrafish retina.

James M. Morrow; Savo Lazic; Belinda S. W. Chang

The visual pigment rhodopsin (rh1) constitutes the first step in the sensory transduction cascade in the rod photoreceptors of the vertebrate eye, forming the basis of vision at low light levels. In most vertebrates, rhodopsin is a single-copy gene whose function in rod photoreceptors is highly conserved. We found evidence for a second rhodopsin-like gene (rh1-2) in the zebrafish genome. This novel gene was not the product of a zebrafish-specific gene duplication event and contains a number of unique amino acid substitutions. Despite these differences, expression of rh1-2 in vitro yielded a protein that not only bound chromophore, producing an absorption spectrum in the visible range (λmax ≈ 500 nm), but also activated in response to light. Unlike rh1, rh1-2 is not expressed during the first 4 days of embryonic development; it is expressed in the retina of adult fish but not the brain or muscle. Similar rh1-2 sequences were found in two other Danio species, as well as a more distantly related cyprinid, Epalzeorhynchos bicolor. While sequences were only identified in cyprinid fish, phylogenetic analyses suggest an older origin for this gene family. Our study suggests that rh1-2 is a functional opsin gene that is expressed in the retina later in development. The discovery of a new previously uncharacterized opsin gene in zebrafish retina is surprising given its status as a model system for studies of vertebrate vision and visual development.


Proceedings of the National Academy of Sciences of the United States of America | 2016

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake

Ryan K. Schott; Johannes Müller; Clement G. Y. Yang; Nihar Bhattacharyya; Natalie Chan; Mengshu Xu; James M. Morrow; Ana-Hermina Ghenu; Ellis R. Loew; Vincent Tropepe; Belinda S. W. Chang

Significance This study provides compelling evidence that the previously reported all-cone retina of a diurnal garter snake in fact contains a population of rod photoreceptors with the appearance, and presumably function, of cones. Our results suggest that the evolution of all-cone retinas occurred not through loss of rods but rather via the evolutionary transmutation of ancestral rods into more “cone-like” photoreceptors, to regain functionality that was lost during the early, possibly fossorial, origin of snakes. This study provides a better understanding of the process by which complex molecular/cellular structures and tissue types can evolve, and how, particularly for sensory systems, physiological constraints can be shaped by selective forces to produce evolutionary novelty. Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the “transmutation” theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single “cones.” Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.


Plasmid | 2010

The p1D4-hrGFP II expression vector: a tool for expressing and purifying visual pigments and other G protein-coupled receptors.

James M. Morrow; Belinda S. W. Chang

The heterologous expression of membrane proteins such as G protein-coupled receptors can be a notoriously difficult task. We have engineered an expression vector, p1D4-hrGFP II, in order to efficiently express visual pigments in mammalian cell culture. This expression vector is based on pIRES-hrGFP II (Stratagene), with the addition of a C-terminal 1D4 epitope tag for immunoblotting and immunoaffinity purification. This vector employs the CMV promoter and hrGFP II, a co-translated reporter gene. We measured the effectiveness of pIRES-hrGFP II in expressing bovine rhodopsin, and showed a 3.9- to 5.7-fold increase in expression as measured by absorbance spectroscopy as compared with the pMT vector, a common choice for visual pigment expression. We then expressed zebrafish RH2-1 using p1D4-hrGFP II in order to assess its utility in expressing cone opsins, known to be less stable and more difficult to express than bovine rhodopsin. We show a λ(280)/λ(MAX) value of 3.3, one third of that reported in previous studies, suggesting increased expression levels and decreased levels of misfolded, non-functional visual pigment. Finally, we monitored HEK293T cell growth following transfection with pIRES-hrGFP II using fluorescence microscopy to illustrate the benefits of having a co-translated reporter during heterologous expression studies.


Evolution | 2015

SWS2 visual pigment evolution as a test of historically contingent patterns of plumage color evolution in warblers

Natasha I. Bloch; James M. Morrow; Belinda S. W. Chang; Trevor D. Price

Distantly related clades that occupy similar environments may differ due to the lasting imprint of their ancestors—historical contingency. The New World warblers (Parulidae) and Old World warblers (Phylloscopidae) are ecologically similar clades that differ strikingly in plumage coloration. We studied genetic and functional evolution of the short‐wavelength‐sensitive visual pigments (SWS2 and SWS1) to ask if altered color perception could contribute to the plumage color differences between clades. We show SWS2 is short‐wavelength shifted in birds that occupy open environments, such as finches, compared to those in closed environments, including warblers. Phylogenetic reconstructions indicate New World warblers were derived from a finch‐like form that colonized from the Old World 15–20 Ma. During this process, the SWS2 gene accumulated six substitutions in branches leading to New World warblers, inviting the hypothesis that passage through a finch‐like ancestor resulted in SWS2 evolution. In fact, we show spectral tuning remained similar across warblers as well as the finch ancestor. Results reject the hypothesis of historical contingency based on opsin spectral tuning, but point to evolution of other aspects of visual pigment function. Using the approach outlined here, historical contingency becomes a generally testable theory in systems where genotype and phenotype can be connected.


Visual Neuroscience | 2012

Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal

Constanze Bickelmann; James M. Morrow; Johannes Müller; Belinda S. W. Chang

Monotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λ(max) of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min-1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min-1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.


Evolution | 2015

The molecular origin and evolution of dim-light vision in mammals.

Constanze Bickelmann; James M. Morrow; Jing Du; Ryan K. Schott; Ilke van Hazel; Steve Lim; Johannes Müller; Belinda S. W. Chang

The nocturnal origin of mammals is a longstanding hypothesis that is considered instrumental for the evolution of endothermy, a potential key innovation in this successful clade. This hypothesis is primarily based on indirect anatomical inference from fossils. Here, we reconstruct the evolutionary history of rhodopsin—the vertebrate visual pigment mediating the first step in phototransduction at low‐light levels—via codon‐based model tests for selection, combined with gene resurrection methods that allow for the study of ancient proteins. Rhodopsin coding sequences were reconstructed for three key nodes: Amniota, Mammalia, and Theria. When expressed in vitro, all sequences generated stable visual pigments with λMAX values similar to the well‐studied bovine rhodopsin. Retinal release rates of mammalian and therian ancestral rhodopsins, measured via fluorescence spectroscopy, were significantly slower than those of the amniote ancestor, indicating altered molecular function possibly related to nocturnality. Positive selection along the therian branch suggests adaptive evolution in rhodopsin concurrent with therian ecological diversification events during the Mesozoic that allowed for an exploration of the environment at varying light levels.


The Journal of Experimental Biology | 2017

A second visual rhodopsin gene, rh1-2, is expressed in zebrafish photoreceptors and found in other ray-finned fishes

James M. Morrow; Savo Lazic; Monica Dixon Fox; Claire Kuo; Ryan K. Schott; Eduardo de A. Gutierrez; Francesco Santini; Vincent Tropepe; Belinda S. W. Chang

ABSTRACT Rhodopsin (rh1) is the visual pigment expressed in rod photoreceptors of vertebrates that is responsible for initiating the critical first step of dim-light vision. Rhodopsin is usually a single copy gene; however, we previously discovered a novel rhodopsin-like gene expressed in the zebrafish retina, rh1-2, which we identified as a functional photosensitive pigment that binds 11-cis retinal and activates in response to light. Here, we localized expression of rh1-2 in the zebrafish retina to a subset of peripheral photoreceptor cells, which indicates a partially overlapping expression pattern with rh1. We also expressed, purified and characterized Rh1-2, including investigation of the stability of the biologically active intermediate. Using fluorescence spectroscopy, we found the half-life of the rate of retinal release of Rh1-2 following photoactivation to be more similar to that of the visual pigment rhodopsin than to the non-visual pigment exo-rhodopsin (exorh), which releases retinal around 5 times faster. Phylogenetic and molecular evolutionary analyses show that rh1-2 has ancient origins within teleost fishes, is under similar selective pressure to rh1, and likely experienced a burst of positive selection following its duplication and divergence from rh1. These findings indicate that rh1-2 is another functional visual rhodopsin gene, which contradicts the prevailing notion that visual rhodopsin is primarily found as a single copy gene within ray-finned fishes. The reasons for retention of this duplicate gene, as well as possible functional consequences for the visual system, are discussed. Summary: An ancient duplication of the rhodopsin gene, rh1-2, is a functional visual pigment and is expressed in retinal photoreceptors.


Proceedings of the National Academy of Sciences of the United States of America | 2017

Evolution of nonspectral rhodopsin function at high altitudes

Gianni M. Castiglione; Frances E. Hauser; Brian S. Liao; Nathan K. Lujan; Alexander Van Nynatten; James M. Morrow; Ryan K. Schott; Nihar Bhattacharyya; Sarah Z. Dungan; Belinda S. W. Chang

Significance Protein evolution in response to different environments has long been of interest to both evolutionary biologists and biochemists. High-altitude specialist catfishes in the Andes mountains offer an opportunity to examine the molecular adaptations accompanying adaptation to cold environments. Rhodopsins and other visual pigments form the first step in vision and have long been a model system for studying the molecular basis of sensory adaptations; however, many of these studies have focused solely on spectral shifts. Recent studies suggest that other aspects of function are as important for visual performance. We demonstrate that high-altitude amino acid variants significantly accelerate RH1 kinetics. These results suggest that the activity–stability trade-off characterized in cold-adapted enzymes also affects adaptation of signaling proteins through similar molecular mechanisms. High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates—properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.


Scientific Reports | 2016

Modulation of thermal noise and spectral sensitivity in Lake Baikal cottoid fish rhodopsins

Hoi Ling Luk; Nihar Bhattacharyya; Fabio Montisci; James M. Morrow; Federico Melaccio; Akimori Wada; Mudi Sheves; Francesca Fanelli; Belinda S. W. Chang; Massimo Olivucci

Lake Baikal is the deepest and one of the most ancient lakes in the world. Its unique ecology has resulted in the colonization of a diversity of depth habitats by a unique fauna that includes a group of teleost fish of the sub-order Cottoidei. This relatively recent radiation of cottoid fishes shows a gradual blue-shift in the wavelength of the absorption maximum of their visual pigments with increasing habitat depth. Here we combine homology modeling and quantum chemical calculations with experimental in vitro measurements of rhodopsins to investigate dim-light adaptation. The calculations, which were able to reproduce the trend of observed absorption maxima in both A1 and A2 rhodopsins, reveal a Barlow-type relationship between the absorption maxima and the thermal isomerization rate suggesting a link between the observed blue-shift and a thermal noise decrease. A Nakanishi point-charge analysis of the electrostatic effects of non-conserved and conserved amino acid residues surrounding the rhodopsin chromophore identified both close and distant sites affecting simultaneously spectral tuning and visual sensitivity. We propose that natural variation at these sites modulate both the thermal noise and spectral shifting in Baikal cottoid visual pigments resulting in adaptations that enable vision in deep water light environments.


Protein Science | 2016

A comparative study of rhodopsin function in the great bowerbird (Ptilonorhynchus nuchalis): Spectral tuning and light‐activated kinetics

Ilke van Hazel; Sarah Z. Dungan; Frances E. Hauser; James M. Morrow; John A. Endler; Belinda S. W. Chang

Rhodopsin is the visual pigment responsible for initiating the phototransduction cascade in vertebrate rod photoreceptors. Although well‐characterized in a few model systems, comparative studies of rhodopsin function, particularly for nonmammalian vertebrates are comparatively lacking. Bowerbirds are rare among passerines in possessing a key substitution, D83N, at a site that is otherwise highly conserved among G protein‐coupled receptors. While this substitution is present in some dim‐light adapted vertebrates, often accompanying another unusual substitution, A292S, its functional relevance in birds is uncertain. To investigate functional effects associated with these two substitutions, we use the rhodopsin gene from the great bowerbird (Ptilonorhynchus nuchalis) as a background for site‐directed mutagenesis, in vitro expression and functional characterization. We also mutated these sites in two additional rhodopsins that do not naturally possess N83, chicken and bovine, for comparison. Both sites were found to contribute to spectral blue‐shifts, but had opposing effects on kinetic rates. Substitutions at site 83 were found to primarily affect the kinetics of light‐activated rhodopsin, while substitutions at site 292 had a larger impact on spectral tuning. The contribution of substitutions at site 83 to spectral tuning in particular depended on genetic background, but overall, the effects of substitutions were otherwise surprisingly additive, and the magnitudes of functional shifts were roughly similar across all three genetic backgrounds. By employing a comparative approach with multiple species, our study provides new insight into the joint impact of sites 83 and 292 on rhodopsin structure‐function as well as their evolutionary significance for dim‐light vision across vertebrates.

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