Development and Application of New Optogenetic Tools Targeted to Intracellular Compartments
Participants:
1) Peter Hegemann (PI; Humboldt University Berlin), coworker
2) Benjamin Rost (PI; Charité, Berlin), coworker:
Abstract:
Development and Application of New Optogenetic Tools Targeted to Intracellular Compartments
The aim of our proposed work program is the development of novel optogenetic tools that allow investigations on subcellular compartments.
We will combine optogenetic actuators and sensors with targeting motives for synaptic vesicles (SV) or lysosomes. The strategy will be based on our recently published work on optogenetic acidification of SV and lysosomes by organelle-specific expression of the light-driven proton pump Arch3. In order to achieve acute depletion of protons from SV and lysosomes we will use a proton channel derived from Arch3, which has been recently developed by the Hegemann lab by point-directed mutagenesis. For the manipulation of luminal chloride we will implement chloride-conductive channelrhodopsins or light-driven chloride pumps. The tetraspanin synaptophysin or CD63 will serve as targeting sequence for SV or lysosomes, respectively. We will characterize and optimize the optogenetic actuators and sensors for subcellular applications beforehand using biophysical approaches. All constructs will be expressed in autaptic neuronal cell cultures using lentivirus or adeno-associated virus as viral gene transfer systems. Our newly developed tools will allow detailed investigations on the ion homeostasis in the endo-lysosomal compartments in living cells.
We also plan to develop novel tools allowing the induction of long-lasting changes in synaptic strength. Initial constructs will be evaluated in primary neuronal cell cultures and hippocampal slice cultures. Ultimately, we will apply these optogenetic actuators in vivo using adeno-associated viral particles, and demonstrate their function in acute hippocampal slices. Light-triggered induction of synaptic strength in genetically defined neurons will enable in vivo studies on the impact of LTP on learning, memory and behavior.
Logic of interaction & planned interactions with other funded members of the SPP
Peter Hegemann & co-workers: Biophysics of optogenetic actuators – development of novel and refinement of exicisting light-driven actuators for the application in subcellular compartments: Molecular biology; protein isolation; time resolved Uv-Vis; FTIR measurements; electrophysiological characterization in xenopus oocytes & HEK293 cells.
Benjamin Rost & coworkers: Neurobiological application of subcellular optogenetic tools: molecular biology; electrophysiology and live-cell imaging in cell lines, primary neurons and brain slices; immunocytochemistry.
Most relevant publications Rost:
Rost BR, Schneider F, Grauel MK, Wozny C, Bentz C, Blessing A, Rosenmund T, Jentsch TJ, Schmitz D, Hegemann P, and Rosenmund C. 2015. Optogenetic acidification of synaptic vesicles and lysosomes. Nature Neuroscience Advance Online Publication 2015/11/09, doi 10.1038/nn.4161.
Watanabe S, Trimbuch T, Camacho-Perez M, Rost BR, Brokowski B, Sohl-Kielczynski B, Felies A, Davis MW, Rosenmund C, and Jorgensen EM. 2014. Clathrin regenerates synaptic vesicles from endosomes. Nature 515(7526):228-33.
Watanabe S, Rost BR, Camacho-Perez M, Davis MW, Sohl-Kielczynski B, Rosenmund C, and Jorgensen EM. 2013. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504(7479):242-7.
Rost BR, Nicholson P, Ahnert-Hilger G, Rummel A, Rosenmund C, Breustedt J, and Schmitz D. 2011. Activation of metabotropic GABA receptors increases the energy barrier for vesicle fusion. J Cell Sci 124(Pt 18):3066-73.
Rost BR, Breustedt J, Schoenherr A, Grosse G, Ahnert-Hilger G, and Schmitz D. 2010. Autaptic cultures of single hippocampal granule cells of mice and rats. Eur J Neurosci 32(6):939-47.
Most relevant publications Hegemann:
Vogt A, Guo Y, Tsunoda SP, Kateriya S, Elstner M, and Hegemann P. 2015. Conversion of a light-driven proton pump into a light-gated ion channel. Sci Rep 5:16450.
Scheib U, Stehfest K, Gee CE, Korschen HG, Fudim R, Oertner TG, and Hegemann P. 2015. The rhodopsin-guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling. Sci Signal 8(389):rs8.
Gasser C, Taiber S, Yeh CM, Wittig CH, Hegemann P, Ryu S, Wunder F, and Moglich A. 2014. Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase. Proc Natl Acad Sci U S A 111(24):8803-8.
Wietek J, Wiegert JS, Adeishvili N, Schneider F, Watanabe H, Tsunoda SP, Vogt A, Elstner M, Oertner TG, and Hegemann P. 2014. Conversion of channelrhodopsin into a light-gated chloride channel. Science 344(6182):409-12.
Stierl M, Stumpf P, Udwari D, Gueta R, Hagedorn R, Losi A, Gartner W, Petereit L, Efetova M, Schwarzel M, Oertner TG, Nagel G, and Hegemann P. 2011. Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa. J Biol Chem 286(2):1181-8.