Curated Optogenetic Publication Database

Abstract: Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

Abstract: Optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. In the current study, we engineered an optogenetic technique that directly inhibits neurotransmitter release. We used a genetically encoded singlet oxygen generator, miniSOG, to conduct chromophore assisted light inactivation (CALI) of synaptic proteins. Fusions of miniSOG to VAMP2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. In cultured neurons and hippocampal organotypic slices, synaptic release was reduced up to 100%. Such inhibition lasted >1 hr and had minimal effects on membrane electrical properties. When miniSOG-VAMP2 was expressed panneuronally in Caenorhabditis elegans, movement of the worms was reduced after illumination, and paralysis was often observed. The movement of the worms recovered overnight. We name this technique Inhibition of Synapses with CALI (InSynC). InSynC is a powerful way to silence genetically specified synapses with light in a spatially and temporally precise manner.

Abstract: We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mito-miniSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.

Abstract: Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.