Scaling up functional analyses of the G protein-coupled receptor rhodopsin

Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. GPCRs are critical to many biological processes; however, the effect of mutations in GPCR-coding genes on GPCR activation and downstream signaling pathways remains poorly understood. 

Benjamin M. Scott, Steven K. Chen, and colleagues have successfully demonstrated the ability to scale up a yeast fluorescent reporter assay to measure in high throughput the effects of over 1200 (of a possible 6612) missense mutations in human rhodopsin, a GPCR involved in visual transduction. Analysis of the results revealed intriguing properties of mutational tolerance potentially generalizable to other transmembrane proteins. Single mutations in rhodopsin have been identified in many clinically identified disease variants, and comparison of these mutants to functional scores from the screen found many of them to be loss of function, as expected. The results demonstrate the exciting potential for high-throughput systems to link genotype to phenotype and will help inform and predict disease phenotypes for clinical interpretation.

The mutational tolerance of rhodopsin varies across its domains
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Evolution of a novel regulatory mechanism for hypoxia tolerance in electric fishes

Hypoxia, or a lack of oxygen in the tissues, is a significant source of metabolic stress. It is a major component of many human diseases, including various forms of cancer. Remarkably, there are animals that display an impressive capacity to withstand lethal levels of hypoxia, providing models to better understand the hypoxic response in vertebrates. Such is the case for Brachyhypopomus, a genus of weakly electric fish that inhabit the Amazon, one of the most challenging aquatic ecosystems in the world. Dissolved oxygen levels deplete on a seasonal and often daily basis, requiring hypoxia tolerance for prolonged periods of time. 

Ahmed A. Elbassiouny and colleagues have used closely related species’ of Brachyhypopomus displaying a range of hypoxia tolerances to investigate the molecular mechanisms of hypoxia-inducible factors (HIFs) – transcription factors known to coordinate the cellular response to hypoxia in vertebrates. They found that HIF1⍺ from hypoxia-tolerant Brachyhypopomus species displayed higher transactivation in response to hypoxia compared to intolerant species. Furthermore, two SUMO-interacting motifs appeared to facilitate the transactivation. Together with computational selection analyses which showed evidence for positive selection in Brachyhypopomus, they showed that the evolution of HIF1⍺ likely underlies adaptations to hypoxia tolerance for this group of fascinating fish.

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Multiple ecological axes drive molecular evolution of cone opsins in Beloniform fishes

Beloniformes, the order including needlefishes, flying fishes, halfbeaks, and allies, comprise over 200 species occupying a wide array of habitats—from the marine epipelagic zone to tropical rainforest rivers. These fish also exhibit a variety of diets, including piscivory, herbivory, and zooplanktivory. The diversity of these species makes them an excellent model group for studying the relative impact of different ecological axes on the molecular evolution of visual transduction genes. 

Katherine D. Chau and colleagues have investigated how diet and habitat have affected the molecular evolution of Beloniform cone opsins, visual proteins that play a key role in bright light and color vision. Via codon-based clade models of evolution, they identified evidence for positive selection in medium-wavelength opsins for piscivores and long-wavelength opsins for zooplanktivores. Medium-wavelength opsins, sensitive to the visual spectrum’s blue/green region, likely enhance prey fish detection against dark backgrounds. In contrast, long-wavelength opsins (sensitive to red light) are thought to help zooplanktivores detect pigment molecules present in surface-dwelling zooplankton. 

Although marine/freshwater habitat transitions also affect opsin molecular evolution, Chau and colleagues found that diet plays a more critical role in Beloniform visual adaptation. Overall, the study suggests that evolutionary transitions along ecological axes produce complex adaptive interactions that affect selection patterns on genes that underlie vision. 

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Adaptive evolution of nearctic deepwater fish vision: implications for assessing functional variation for conservation

Intraspecific functional variation, the functional variation of a trait within a species, is critical for adaptation to rapidly changing environments. Ciscoes and Deepwater Sculpin are two lineages of North American deep-dwelling fish that recently, less than 15,000 years ago, began to inhabit postglacial lakes differing in water depth, clarity, and composition. As such, they make ideal species for studying intraspecific functional variation and gleam insight into rapid visual evolution. These species are also of cultural, commercial, and nutritional importance to Indigenous communities but have faced significant declines due to environmental perturbations and overfishing, among other factors. Understanding the ecological and genetic factors involved in these declines is essential for their conservation. 

A metabarcoding approach to map functionally relevant rhodopsin variants.

Alex Van Nynatten and colleagues have identified depth-related variation in the dim-light visual pigment rhodopsin that evolved convergently in ciscoes and deepwater sculpins. In vitro characterization of the convergent alleles revealed blue shifts compared with more widely distributed alleles, closely mirroring the visual aquatic environment of the specific fish they were collected from. In collaboration with the Saugeen Ojibway Nation, Van Nynatten et al. have also developed and tested a metabarcoding approach to efficiently and accurately map the functionally relevant rhodopsin variation. These initiatives are highly informative, outlining a framework to apply the study of adaptive molecular evolution to conservation efforts.

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Convergent Evolution of Dim Light Vision in Owls and Deep-Diving Whales

Rhodopsin light activation produces all-trans retinal, a toxic byproduct. As a result, animals with enhanced dim light sensitivity are at higher risk of retinal degeneration when exposed to bright light conditions. Rod arrestin (Arr-1) binds to rhodopsin and promotes sequestration of excess all-trans retinal, potentially protecting against photoreceptor cell death.

Recently, Castiglione et al. have discovered a rare rod arrestin (Arr-1) mutation in owls and deep-diving whales that significantly enhances the sequestration of all-trans retinal in vitro. This exciting functional convergence in animals at risk of retinal damage suggests Arr-1’s underappreciated role in the photoprotection of the eye and has important implications for visual disease.

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Chang Lab and colleagues receive Human Frontier Science Program grant to pursue studies into the evolution of the multifunction rhodopsin protein

We are excited to announce that our lab has received a $2M grant to study the multifunctional role of rhodopsin! The research will be conducted as a collaborative effort with the Feuda lab at the University of Leicester, the Menon lab at Cornell University, and the Göpfert lab at the University of Göttingen. Check out this great article from the CSB department at UofT for more.  

Opsins are a class of proteins commonly known to function as visual pigments essential for eye vision. Research over the past decade has expanded this view, implicating their role in detecting other sensory stimuli such as heat, touch and taste in various species. Remarkably, rhodopsin Rh1, the most commonly studied opsin, can also scramble proteins inside-out at the cell membrane, suggesting a role extending beyond the senses. 

With funding from the Human Frontiers Science Program, the labs will target the Rh1 opsin in fruit flies to understand how the protein’s multiple functions constrain its sequence variation. Rhodopsin mutants that fold properly but are not present in nature will be synthesized in flies to determine whether they might gain extraordinary abilities in sensorial detection and other activities.

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Whales Evolved Swiftly to Become Deep Divers

Awesome news article from the CSB department at UofT highlighting a recent PNAS paper from the Chang lab, featuring the work of former PhD student Sarah Dungan!

Many cetacean species can dive to extraordinary depths on a single breath, but the evolutionary origins of deep-sea foraging in ancestral cetaceans remain unclear. In their publication, they present a resurrected ancestral cetacean visual protein (rhodopsin).

Their findings suggest that ancient whales were active at mesopelagic depths and had evolved a faster dark adaptation rate, a trait that allows diving mammals to rapidly adjust to dimming light. Their results also indicate that the ancestor of modern cetaceans was a deeper diver.

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This Fish Has Adapted to Canada’s Deepest Coldest Lakes. UTSC Researchers are Unravelling it’s Genetics to Find Out How.

Great article from UTSC covering the incredible work Alex Van Nynatten, a postdoc in the Lovejoy lab, has been doing with the Deepwater Sculpin in collaboration with the Chang lab! This fish is quite impressive as they are known to live almost exclusively in lakes with temperatures below 8 °C, and at depths greater than 35 metres.

The entire genome of this fascinating species is currently being sequenced to see how this otherwise unassuming fish has adapted to such harsh conditions. Alex has special interest in in studying the fish’s vision genes and their adaptations to this cold and dark environment.

Alex Van Nynatten, a postdoc in the Lovejoy lab.
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Convergent patterns of evolution of mitochondrial oxidative phosphorylation (OXPHOS) genes in electric fishes

The ability to generate and detect electric fields is vital for the survival of several groups of fishes. Authors Ahmed A. Elbassiouny, Nathan R. Lovejoy and Belinda S.W. Chang speculated that electric fish may be able to meet the high metabolic demands of bioelectrogenesis, as a result of the adaptive evolution of genes encoding for the mitochondrial OXPHOS cellular machinery. Evidence for this was found in two independently derived clades of weakly electric fishes, South American Gymnotiformes and African Mormyroidea.

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Screening of Chemical Libraries Using a Yeast Model of Retinal Disease

Pathogenic mutations cause rhodopsin to misfold and disrupt its function. In this study, a yeast-based assay was used to screen for compounds that have the potential to rescue the function of mutant rhodopsin. It was confirmed that 9-cis retinal could partially rescue light-dependent activation of disease-associated rhodopsin mutation (P23H). A phenotypic screen was also done with yeast assays to screen compounds from the LOPAC1280 library and a peptidomimetic library.

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