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

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    Author
    Godwin, Walton C.; Hoffmann, George F.; Gray, Taylor J.; Hughes, Robert M.
    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 β 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.
    URI
    http://hdl.handle.net/10342/7577
    Subject
     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 
    Date
    2019-10-03
    Citation:
    APA:
    Godwin, Walton C., & Hoffmann, George F., & Gray, Taylor J., & Hughes, Robert M.. (October 2019). Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools. Journal of Biological Chemistry, (294:45), p.16918-16929. Retrieved from http://hdl.handle.net/10342/7577

    Display/Hide MLA, Chicago and APA citation formats.

    MLA:
    Godwin, Walton C., and Hoffmann, George F., and Gray, Taylor J., and Hughes, Robert M.. "Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools". Journal of Biological Chemistry. 294:45. (16918-16929.), October 2019. April 20, 2021. http://hdl.handle.net/10342/7577.
    Chicago:
    Godwin, Walton C. and Hoffmann, George F. and Gray, Taylor J. and Hughes, Robert M., "Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools," Journal of Biological Chemistry 294, no. 45 (October 2019), http://hdl.handle.net/10342/7577 (accessed April 20, 2021).
    AMA:
    Godwin, Walton C., Hoffmann, George F., Gray, Taylor J., Hughes, Robert M.. Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools. Journal of Biological Chemistry. October 2019; 294(45) 16918-16929. http://hdl.handle.net/10342/7577. Accessed April 20, 2021.
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