Optogenetic stimulation of intracellular Ca2+ release from the endoplasmatic reticulum
1) Alexander Gottschalk (PI; University of Frankfurt), coworker:
2) Stefan Lehnart (PI; University of Göttingen), coworker: Miroslav Dura
3) Philipp Sasse (PI; University of Bonn), coworker: Tobias Bruegmann
Optogenetic stimulation of intracellular Ca2+ release from the endoplasmic reticulum
Optogenetic tools can be generated by combination of exogenous, light switchable proteins with (endogenous) effector proteins (‘combined optogenetics', ‘two-component optogenetics'). The aim of this consortium of three laboratories of complementary expertise is to directly modulate the essential process of Ca2+ release from intracellular stores by optogenetic tools. We thus want to generate and deploy new optogenetic hybrid proteins. In a first approach, we fused the endogenous (Ca2+-activated) Ca2+ release channel (ryanodine receptor, RyR) in the roundworm Caenorhabditis elegans with a Ca2+-conducting variant of a light-switchable cation channel (‘CatCh'). Preliminary work using the ‘optoRyR’ fusion protein is promising: Illumination led to altered locomotion in C. elegans expressing optoRyR.
In the proposed project, we will thoroughly characterize optogenetic tools for Ca2+ release, develop them further and transfer them to mammalian cells. To this end, additional optoRyR variants and alternative hybrid constructs will be generated and examined by worm behavioral experiments, and by electrophysiological as well as Ca2+ measurements in C. elegans, in HEK293 cells and in cardiac myocytes. Direct biophysical single-channel measurements of the isolated proteins in lipid bilayer experiments will serve for precise determination of the function of optoRyR variants and to decrypt the mechanism of Ca2+-release (i.e. amplification of Ca2+ release through RyR vs. mechanically-induced RyR opening). Since signal amplification by RyR depends on the formation of larger protein networks (RyR clusters), optoRyR clusters are analyzed by super-resolution STED microscopy and compared with the behavior of endogenous RyR2 clusters. For functional investigation of RyR-dependent cardiac arrhythmias, which depend on excessive Ca2+ release (i.e. Catecholaminergic Polymorphic Ventricular Tachycardia - CPVT) the optoRyR proteins will be used in two surrogate models: in the C. elegans pharynx in vivo and in three-dimensional cardiac muscle, differentiated from optoRyR-expressing stem cells.
We expect that optoRyR will enable a very high spatial and temporal precision of Ca2+ release and will thus have substantial advantages over the standard, diffusion-limited pharmacological RyR modulation. Since Ca2+ release from intracellular stores is essentially involved in the physiology and pathology of a variety of cell types, as well as in the function of neurons, the new optoRyR proteins should have a very broad range of applications and will enable entirely novel mechanistic studies of acquired or inherited diseases (e.g. heart failure, arrhythmia, muscle weakness or diabetes mellitus), as well as of synaptic transmission.
Logic of interactions:
OptoRyR constructs will be generated for C. elegans and tested by the Gottschalk group, and in parallel, by the Sasse group, for the mouse. The Lehnart group will provide essential biophysical experiments (single-channel measurements) to characterize the mechanism of action of the generated constructs.
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