Mechanism, engineering and application of Rhodopsin-guanylyl cyclases
Participants:
1) Herwig Baier (PI, Max Planck Institute of Neurobiology, Munich), coworker: Miguel Fernandes
2) Marcus Elstner (PI; Karlsruhe Institute of Technology, KIT), coworker:
3) Peter Hegemann (PI; Humboldt University Berlin), coworker: Ulrike Scheib
4) Franziska Schneider (PI; University of Freiburg), coworker: Ramona Kopton
Abstract:
Mechanism, engineering and application of Rhodopsin-guanylyl cyclases
Channelrhodopsin, which was discovered and described as a light-gated ion channel in the laboratory of Peter Hegemann and colleagues, has revolutionized the field of neuroscience over the past decade by enabling researchers to specifically activate selected neurons in a large ensemble of neuronal cells with short light flashes, a technology now known as "Optogenetics." However, though highly desirable, the inactivation of specific cells using moderate or low light intensities is not yet possible. The recently discovered rhodopsin-guanylyl-cyclase (RhGC) of the fungus Blastocladiella emersonii offers an elegant solution to this problem. RhGC is a totally novel and uncharacterized sensory photoreceptor, and the first member of an enzyme rhodopsin family that urgently awaits in-depth characterization. Accordingly, our goals are to obtain a comprehensive understanding of this novel photoreceptor, to determine its utility as an optogenetic actuator, and to begin to explore in vivo application. The project is subdivided into four objectives. The first objective is the detailed characterization of RhGC in cell lines, in neurons and as recombinant protein and engineering of the guanylyl module into an adenylyl cyclase resulting a RhAC. We will coexpress of RhGC or RhAC with a cNG-gated K+ channel to develop a "Light-Hypopolarizer" for cell inactivation. The second objective is to engineer, on the basis of structure-based modeling and QM/MM calculations, the rhodopsin into a photochromic bimodal switchable rhodopsin that is activated with blue light and inactivated with UV-light or vice versa. Third, we will apply the most promising new effector proteins for remote optical control of axon guidance and neuronal activity in the developing zebrafish nervous system. Finally, we will use subcellularly targeted RhGC to study cardiac pacemaker activity and Photo-Hyperpolarizer to analyse electrical conduction in an in vivo model for cardiac injury in rodents. Together, our collaborative work will establish RhGC and its derivatives as a powerful new optogenetic toolkit for cell biology, neuroscience and cardiovascular research.
Logic of interactions:
P.Hegemann: Biochemical and biophysical characterization of Rhodopsin-Cyclases;
M.Elstner: Theory: development of a concept for a photochromic and bimodal switchable Rhodopsins-Cyclases;
H.Baier: Application of RhGC/ACs to Zebrafish for the analysis of signaling pathways,
F.Schneider: Application of RhGC/AC to heart cells
Publications Baier, relevant for the proposal:
Barker A, Baier H (2015). Sensorimotor decision-making in the zebrafish tectum. Curr Biol 25:2804-14.
Kubo F, Hablitzel B, Dal Maschio M, Driever W, Baier H, Arrenberg AB (2014). Functional architecture of an optic flow responsive area that drives horizontal eye movements in zebrafish. Neuron 81:1344-59.
Thiele T, Donovan JC, Baier H (2014). Modular descending control of swim posture in zebrafish. Neuron 83: 679-691.
Arrenberg AB, Stainier DY, Baier H, Huisken J (2010). Optogenetic control of cardiac function. Science 330:971-974.
Arrenberg AB, Del Bene F, Baier H (2009). Optical control of zebrafish behavior with halorhodopsin. Proc Natl Acad Sci U S A 106:17968-17973.
Publications Schneider, relevant for the proposal:
B. R. Rost*, F. Schneider*, M. K. Grauel, C. Wozny, C. Bentz, A. Blessing, T. Rosenmund, T. Jentsch, D. Schmitz, P. Hegemann, and C. Rosenmund, “Optogenetic Acidification of Synaptic Vesicles and Lysosomes,” Nature Neuroscience, advance online publication 09 Nov 2015, doi 10.1038/nn.4161
F. Schneider, C. Grimm and P. Hegemann, “The biophysics of channelrhodopsin”, Annual Review of Biophysics, vol. 44, pp. 167-186, 2015
J. Wietek, J. S. Wiegert, N. Adeishvili, F. Schneider, H. Watanabe, S. P. Tsunoda, A. Vogt, M. Elstner, T. G. Oertner, and P. Hegemann, “Conversion of Channelrhodopsin into a Light-Gated Chloride Channel,” Science, Vol. 344, no. 6182, pp. 409-412, 2014.
F. Schneider, D. Gradmann, and P. Hegemann, “Ion Selectivity and Competition in Channelrhodopsins,” Biophysical Journal, vol. 105, pp. 91-100, July 2013.
M. Prigge*, F. Schneider*, S. P. Tsunoda, C. Shilyansky, J. Wietek, K. Deisseroth, and P. Hegemann, “Color-tuned Channelrhodopsins for Multiwavelength Optogenetics.,” The Journal of Biological Chemistry, 2012. * equal contribution