Phototaxis and Phototransduction Mechanisms in the Model System C. elegans by Alexander Ward A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Neuroscience) in The University of Michigan 2010 Doctoral Committee: Assistant Professor Xian-Zhong Shawn Xu, Chair Associate Professor Jonathan B. Demb Assistant Professor John Kim Assistant Professor Geoffrey G. Murphy Assistant Professor Michael Mark Alexander Sutton © Alexander Ward 2010 Acknowledgements I thank J. Demb, G. Murphy, J. Kim, M. Sutton, S. Xu, P. Hu, A. Kumar and B. Ye for comments; C. Bargmann for providing tax-2 rescuing strains; M. Koelle for a PTX plasmid; B. Decaluwe, M. Xia and S. Gu for technical assistance; L. Kang for movie editing; Q. Liu and Z.W. Wang for assistance in setting up recording; and members of the Xu lab for advice. Some strains were obtained from the Caenorhabditis Genetics Center, the Oklahoma Knockout Consortium and S. Mitani. A.W. was supported by a US National Institutes of Health predoctoral training grant and a predoctoral training grant from the National Eye Institute (T32EY13934 to The University of Michigan). J.G. and Z.X. received support from the Chinese 111 project (CB06081). This work was supported by the US National Institute of General Medical Sciences (NIGMS) and the Pew scholars program (X.Z.S.X.). Contributions: Chapter 2. Alex Ward (A.W.), Jie Liu (J.L.), Zhaoyang Feng (Z.F.), and X.Z. Shawn Xu (X.Z.S.X) were the authors for data in Chapter 2. A.W. and J.L. contributed equally to this work. A.W. conducted the experiments and analyzed the data in Figures 2.1–2.4. J.L. conducted the experiments and analyzed the data in Figures 2.5 and 2.6. Z.F. developed tools to acquire and analyze behavioral data. X.Z.S.X. supervised the project and wrote the manuscript. Chapter 3. Jie Liu (J.L.), Alex Ward (A.W.), Jingwei Gao (J.G.), Yongming Dong (Y.D.), Nana Nishio (N.N.), Hitoshi Inada (H.I.), Lijun Kang (L.K.), Yong Yu (Y.Y.), Di Ma (D.M.), Tao Xu (T.X.), Ikue Mori (I.M.), Zhixiong Xie (Z.X.) and X.Z. Shawn Xu (X.Z.S.X) were the authors for data in Chapter 3. A.W. and J.L. contributed equally to this work. J.L. performed most of the electrophysiological recordings and analyzed the data. A.W. carried out most of the molecular biology, genetic and behavioral experiments and analyzed the data. J.G. and Z.X. performed some of the molecular biology, genetic and behavioral experiments. Y.D. and L.K. carried out some of the recordings. N.N., H.I. and I.M. isolated pde mutants. A.W. and D.M. isolated lite-1 mutants. A.W., Y.Y. and T.X. mapped lite-1 mutants. X.Z.S.X. supervised the project and wrote the paper with help from all of the other authors. ii Table of Contents Acknowledgements……………………………………………………………………...ii List of Figures……………………………………………………………………………v Abstract………………………………………………………………………………….vii Chapter 1. Introduction……………….………………….………………………………1 An overview of phototransduction……………..………………………1 Ciliated photoreceptor cells……………………….…………………...3 Activation of the photoresponse in vertebrate rods and cones…….4 Termination of the photoresponse in vertebrate rods and cones…………………………………………………………..7 Regulation of cGMP production in vertebrate rods and cones……10 Light-dependent adaptation of the photoresponse in vertebrate rods and cones...............................................................................11 Invertebrate GC-based ciliary phototransduction motifs…………..13 Rhabdomeric phototransduction……………………………………..14 Drosophila vision and the compound eye…………………………..15 Activation of the Drosophila photoresponse………………………..17 Termination of the Drosophila photoresponse…………………......21 Light-dependent adaptation in Drosophila…………………………..25 Mammalian intrinsically photosensitive retinal ganglion cells (ipRGCs)………………………………………………………….26 An overview of C. elegans sensory systems……………...………..28 C. elegans chemosensory systems………………………………….30 Chemotransduction molecules in C. elegans……………………….33 C. elegans Mechanosensory systems………………………………37 Mechanotransduction molecules in C. elegans…………………….39 Other C. elegans sensory modalities……………………………......41 C. elegans are photosensitive and utilize a cGMP/CNG-based phototransduction mechanism……….………..42 2. Light-sensitive neurons and channels mediate phototaxis in C. elegans……………………………………………………………….45 Summary…………………………………………………………….….45 Introduction……………………………………………………………..46 Experimental Procedures……………………………………………..47 Results………………………………………………………………….51 Discussion………………………………………………………………67 iii 3. C. elegans phototransduction requires a G protein–dependent cGMP pathway and a taste receptor homolog………………………….72 Summary………………………………………………………………..72 Introduction……………………………………………………………..73 Experimental Procedures………………………………………….....75 Results………………………………………………………………….78 Discussion…………………………………………………………….106 4. Conclusion.........................................................................................111 C. elegans exhibits light-avoidance behavior……………………..113 UV Light evokes inward depolarizing cGMP/CNG-mediated photocurrents in ASJ and ASK……………………..………………115 Light activates a G protein-dependent pathway which requires membrane guanylate cyclases……….…………………..117 The taste receptor-like protein LITE-1 is required for light responses and functions upstream of G protein signaling……....119 Future Directions……………………………………………………..120 Bibliography…………………………………………………………………………..125 iv List of Figures Figure 1.1 Schematic of rod phototransduction……………………………………………...5 1.2 Schematic of Drosophila phototransduction …………………………………..17 1.3 Schematic of C. elegans phototransduction …………………………………..43 2.1 Light evokes avoidance responses in C. elegans in a dose-dependent manner……………………………………………….......52 2.2 Behavioral quantification of phototactic responses………………………......55 2.3 Prolonged light exposure induces paralysis/lethality in worms………….......57 2.4 Phototaxis in C. elegans requires ciliary sensory neurons………………......59 2.5 Light stimulates the photoreceptor neuron ASJ by evoking an inward current carried by CNG channels………………………………......62 2.6 The light-sensitive CNG channels in the photoreceptor neuron ASJ are sensitive to cGMP……………………………………………………………66 3.1 Phototransduction in ASJ is a G protein–mediated process…………………81 3.2 Phototransduction in ASJ requires membrane-associated guanylate cyclases……………………………………………………………….86 3.3 Guanylate cyclases act downstream of G proteins and upstream of CNG channels to mediate phototransduction…………………..90 3.4 Light, GTPγS and cGMP activate the same type of CNG channels in photoreceptor cells…………………………………………………………….92 3.5 LITE-1 is required for phototransduction in photoreceptor cells…………….97 3.6 LITE-1 functions upstream of G proteins……………………………………..100 3.7 Transgenic expression of LITE-1 can confer photo-sensitivity to the photo-insensitive neuron ASI…………………………………………..106 4.1 Schematic of C. elegans photoreceptor connectivity………………………..114 Supplementary Figure 2.1 Worms respond to light in a duration-dependent manner……………………53 2.2 The threshold of UV-A light intensity in inducing an avoidance response in worms…………………………………………………...…………..56 2.3 Additional laser ablation data……………………………………………………58 2.4 ASJ is more sensitive to UV light than to violet, blue and green light…...….61 2.5 The inhibitory effect of L-cis-diltiazem on the light- and cGMP-induced currents is reversible………………………………………..…64 2.6 Schematic models……………………………………………………………..…69 3.1 gpa-1, gpa-3 and goa-1 single mutants do not show a noticeable defect in phototransduction in ASJ……………………………………………..83 v 3.2 Rescue of odr-1 phototransduction defect……………………………………..89 3.3 Supplement of cGMP does not restore photosensitivity in ASJ of daf-11 mutant worms…………………………………………………….93 3.4 Dendrogram of C. elegans taste receptors…………………………………….95 3.5 LITE-1 is also required for phototransduction in the photoreceptor cell ASK……………………………………………………………………………96 3.6 The photoreceptor cell ASK requires CNG channels and membrane-associated GCs but not PDEs for phototransduction…………...99 3.7 The light-gated channel channelrhodopsin-2 (ChR2) is functional in lite-1, daf-11, odr-1, tax-2 and tax-4 mutant worms………………………102 3.8 Reactive oxygen species (ROS) evoke an inward current in ASJ independently of LITE-1………………………………………………………104 3.9 A model for the C. elegans phototransduction cascade in the photoreceptor cell ASJ………………………………………………………….107 vi Abstract Phototaxis and Phototransduction Mechanisms in the Model System C. elegans By Alexander Ward Chair: Xian-Zhong Shawn Xu C. elegans has become an increasingly popular model system for the study of sensory systems, in particular olfactory transduction and mechanotransduction. However, C. elegans is eyeless and lives in darkness (i.e. soil), and this organism has generally been presumed to be photoinsensitive. The ability to sense light is crucial to the survival of many organisms. In my thesis work I challenged the assumption that C. elegans is photoinsensitive, reasoning that light might serve functions other than “vision” per se. For instance, negative phototaxis behavior in C. elegans could function to retain worms in soil, or protect them from harmful effects of UV light. In my thesis research, I found that light stimuli, indeed, elicit avoidance behavior in C. elegans, and that prolonged light stimulation is lethal to worms. We also identified a group of ciliary sensory neurons as candidate photoreceptor cells. In a subset of these neurons (ASJ and ASK), we showed that light evokes a depolarizing conductance mediated