Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)
Walton C. Godwin (Creator)
Taylor J. Gray (Creator)
George F. Hoffmann (Creator)
Robert M. Hughes (Creator)
East Carolina University (ECU )
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Abstract: Creation of optogenetic switches for specific activation of cell death pathways can provide insights into apoptosis and could also form a basis for noninvasive , next-generation therapeutic strategies. Previous work has demonstrated that cryptochrome 2 (Cry2)/cryptochrome-interacting ß helix-loop-helix (CIB) , a blue light-activated protein-protein dimerization module from the plant Arabidopsis thaliana , together with BCL2-associated X apoptosis regulator (BAX) , an outer mitochondrial membrane-targeting pro-apoptotic protein , can be used for light-mediated initiation of mitochondrial outer membrane permeabilization (MOMP) and downstream apoptosis. In this work , we further developed the original light-activated Cry2-BAX system (hereafter referred to as OptoBAX) by improving the photophysical properties and light-independent interactions of this optogenetic switch. The resulting optogenetic constructs significantly reduced the frequency of light exposure required for membrane permeabilization activation and also decreased dark-state cytotoxicity. We used OptoBAX in a series of experiments in Neuro-2a and HEK293T cells to measure the timing of the dramatic morphological and biochemical changes occurring in cells after light-induced MOMP. In these experiments , we used OptoBAX in tandem with fluorescent reporters to image key events in early apoptosis , including membrane inversion , caspase cleavage , and actin redistribution. We then used these data to construct a timeline of biochemical and morphological events in early apoptosis , demonstrating a direct link between MOMP-induced redistribution of actin and apoptosis progression. In summary , we created a next-generation Cry2/CIB-BAX system requiring less frequent light stimulation and established a timeline of critical apoptotic events , providing detailed insights into key steps in early apoptosis.

Additional Information

Language: English
Date: 2019
optogenetics, actin, cryptochrome, cytoskeleton, Bax, apoptosis, mitochondrial apoptosis, BCL2-associated X apoptosis regulator (BAX), cytoskeletal dynamics, effector molecule, mitochondrial outer membrane permeabilization (MOMP), OptoBAX 2.0 system

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