Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and <em>Xenopus</em> Embryonic Development

Abstract

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Exercise Improves Endothelial Function Associated with Alleviated Inflammation and Oxidative Stress of Perivascular Adipose Tissue in Type 2 Diabetic Mice

“…Culture of Primary Aorta Endothelial Cells. The primary endothelial cells were isolated and cultured from the exercised and nonexercised db/db mice (n = 8/group) with slight modification [22]. In brief, the thoracic aorta was quickly removed and put it in ice-cold 1x PBS (sterile), then was gently flushed with ice-cold PBS to remove the blood, and the attached adipose tissue was removed.…”

Induction of Signal Transduction by Using Non‐Channelrhodopsin‐Type Optogenetic Tools

“…Currently,s everal optogenetic tools have been appliedb oth to cultured cells and to in vivo studies, including in Drosophila, [73b, 81] in Xenopus embryos, [82] in zebrafishe mbryos, [68b] and in mice. [12, 41, 62, 66b] Further improvement of in vivo studies might be facilitated by using red-and far-red-light-responsive photoswitches because these light wavelengths can penetrate thick tissues.H owever,t here are several issues associatedw ith the use of the PhyB-PIF3 and BphP1-PpsR2 systems, as described above.A dditionally,p hycocyanobilin as ac hromophorem ust be artificially added to the PhyB system because phycocyanobilin is absenti nm ammalian cells, although one recent study improved the utility of this optotool in cultured cells by expressing the enzymes responsible for synthesizing phycocyanobilin.…”

Optogenetic Application to Investigating Cell Behavior and Neurological Disease

“…Finally, optogenetics can function as a precision perturbation tool in developmental biology (Krueger et al, 2019). In vivo application include decoding signaling dynamics during Xenopus development, where light-mediated Raf/MEK/ERK activation, accomplished by membrane localization of Raf1 via CRY2-CIBN association, was demonstrated to induce a tail-like structure after germ layer formation (Krishnamurthy et al, 2016(Krishnamurthy et al, , 2017, and optoWnt activation results in axis duplication (Krishnamurthy et al, 2021).…”