Functional Characterization and Molecular Evolutionary Analyses of Rhodopsin in Fishes and Other Vertebrates
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Functional Characterization and Molecular Evolutionary Analyses of Rhodopsin in Fishes and other Vertebrates by Gianni Marcello Castiglione A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Cell and Systems Biology University of Toronto © Copyright by Gianni Marcello Castiglione 2017 Functional Characterization and Molecular Evolutionary Analyses of Rhodopsin in Fishes and other Vertebrates A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Cell and Systems Biology University of Toronto © Copyright by Gianni Marcello Castiglione 2017 Abstract The visual perception of light is complex, achieved through sophisticated biological systems ranging from the molecular interactions of visual system proteins, to computational processing within retinal neuronal circuits, to the higher cortical synthesis of visual inputs. Photon absorption by the rod visual pigment rhodopsin forms the first step in the visual transduction cascade, ultimately mediating all dim-light organismal visual sensitivity. Natural selection has moulded vertebrate rhodopsin into a remarkable molecular machine, with a stunning sensitivity to single photons and an ultra-fast, femtosecond photoproduct formation. These evolutionary innovations are underpinned by the elegant structural and functional features of rhodopsin, serving as the basis for our understandings of other class A G protein-coupled receptors. Much remains to be discovered of the precise molecular mechanisms which govern rhodopsin structure and function, with mutational epistasis and other features of protein science presenting a formidable analytical barrier against progress. Comparative evolutionary approaches investigating protein adaptation are a powerful yet underexplored means by which novel features of protein science can be elucidated. In this thesis, an interdisciplinary approach involving computational models of molecular evolution combined with protein modelling and in vitro ii experimental tests of function were employed towards unravelling how natural variation in rhodopsin can reflect evolutionary adaptations that can ultimately illuminate and predict novel features of rhodopsin structure and function. Following a general introduction, Chapter II presents the first evidence for the cold-adaptation of rhodopsin, using high altitude Andean catfishes (Siluriformes) as a model system. Molecular targets under selection at high altitudes are shown to be highly similar between high altitude Tibetan Plateau and Andean catfish rhodopsins, with evidence in Chapter III suggesting possible phenotypic convergence of rhodopsin kinetics at high altitudes. In Chapter IV a new functional feature of rhodopsin was discovered, comprising a compensatory structural unit which modulates both rhodopsin spectral sensitivity and light- activated kinetics. This thesis identifies several novel features of rhodopsin structure and function while presenting case studies of protein adaptation. This multidimensional explication reflects and contributes to the emerging understanding that protein structure and function is inextricably linked to shifts in ecology, environment and the dynamics of molecular evolution. iii Acknowledgements “Of a human’s life, their time is a point, their existence a flux, their sensation clouded, their body’s entire composition corruptible, their vital spirit an eddy of breath, their fortune hard to predict, their fame uncertain. Briefly, all the things of the body, a river; all the things of the spirit, dream and delirium; their life a warfare and a sojourn in a strange land, their after-fame oblivion. What then can be their escort through life? One thing and one thing only, Philosophy” -Marcus Aurelius Antoninus, written among the Quadi on the river Gran. I c. 170 A.D. In 2016, such an utterance is regarded as scandalous and outdated, the providence of old men with no “evidence”. The common thread throughout 2500 years of western thought is the idea of nature as an infinite source of wisdom that we, strangely, can access. The research approaches taken in this thesis were motivated out of a deference to that same commanding elegance that Aurelius and countless other philosophers noticed, and often agonized over. I have certainly agonized over this thesis: it has been damn hard work with way too much of a political element for an idealist like myself. Insofar as my endurance has been tested, it remains light years away from that demonstrated by my grandparents, who from Racalmuto, Sicilia and 中國 廣東省 台山 端芬大新里 台城 那金吉昌里村, worked harder than I can ever comprehend. All so their descendants like me could waste time quoting Marcus Aurelius! I wish I could thank you all in person. Your willpower is a constant source of inspiration. Parallel to my graduate efforts was the long, extrinsic emotional distress my partner Lina and I navigated, wherein I learned the exact definition of fortitude and perseverance. The difficulty of those years makes the completion of this thesis all the more satisfying. The recognition may be subtler, but the resolution initiated by Lina is more laudable than all the work represented in this thesis. I love her more than words can describe. Our giant baby Makwa the Malamute remains the living proof that scientific reductionism is a woefully inadequate model of reality, and I couldn’t love him more for it. I am indebted to my mother and father for instilling in me a sense of curiosity for biology and complexity at a young age, and for their support in getting me to where I am today. I hope you both know that I am proud to be your son. I owe thanks to my siblings as well, for they constantly put me in my place, an absolutely invaluable contribution done with love and care. Thanks to my supervisor Dr. Belinda Chang, for the opportunity to work in her laboratory and for teaching me how to transform creative energies into concrete achievements. And of course, thanks to my labmates for their advice and support over the years, especially Frances, Nihar, and Ryan, and maybe Amir. What a strange and rewarding time it has been. iv Table of Contents Title page……………………………………………………………………………..……………i Abstract……………………………………………………………………………………………ii Acknowledgements………………………………………………………………………….……iv Table of Contents…………………………………………………………………………………v List of Figures………...…………………………………………………………………………..ix List of Tables..………….…………………………………………………………………………xi List of Abbreviations……………………………………………………………………………xiii Author Contributions…………………………………………………………………...……….xvi Chapter I General Introduction ....................................................................................................... 1 1.1. The Visual System ........................................................................................................... 2 1.1.1 Evolution of the vertebrate Visual System ............................................................... 2 1.1.2 The Retina and Photoreceptors ................................................................................. 4 1.1.3 Visual Pigments and Phototransduction ................................................................... 6 1.1.4 Rhodopsin Structure.................................................................................................. 7 1.1.5 Rhodopsin Light Activation .................................................................................... 11 1.1.6 Rhodopsin Thermal Activation ............................................................................... 12 1.1.7 Natural Variation and Adaptation in Rhodopsin .................................................... 14 1.2. Fish Systems ................................................................................................................... 16 1.2.1 Visual Ecology of Fishes ........................................................................................ 16 1.2.2 Otophysi .................................................................................................................. 18 1.2.3 Siluriformes............................................................................................................. 18 1.2.4 Andean Siluriformes ............................................................................................... 20 1.2.5 Tibetan Plateau Siluriformes................................................................................... 21 1.2.6 Siluriform Vision .................................................................................................... 22 1.3. Molecular Evolution ....................................................................................................... 24 1.3.1 Theory ..................................................................................................................... 24 1.3.2 Molecular Evolution of Proteins and Their Coding Sequences .............................. 25 1.3.3 Computational Estimates of dN/dS .......................................................................... 26 1.3.4 Using dN /dS to Identify Instances of Adaptive Evolution ...................................... 27 1.4. Thesis Objectives ........................................................................................................... 29 v 1.5. Thesis Overview ............................................................................................................. 31 1.6. Figures ...........................................................................................................................