Name: light-mediated reversible modulation of the mitogen-activated protein kinase pathway during cell differentiation and xenopus embryonic development
Uploaded: 2017-06-15T15:00:00
Description: Watch this Scientific Journal Video about Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development at JoVE.com

This work was supported by the University of Illinois at Urbana-Champaign (UIUC) and the National Institutes of Health (NIGMS R01GM111816).

Animal research was conducted in accordance with guidelines set by the Illinois Institutional Animal Care and Use Committee (IACUC) and the University of Illinois Department of Animal Resources (DAR).
1. Optogenetic Induction of Protein Localization in BHK21 Mammalian Cell Culture
NOTE: Steps 1.1-1.3 provide a method to assemble a cell culture chamber for imaging with high-magnification objectives (e.g., 63X or 100X), which typically have short working distan...

When building the light box, the power of individual LEDs should be measured. Based on previous experience, the power output can vary between individual LEDs due to manufacturing variance. Select a set of LEDs that have a power output within 10% of each other. The number of LEDs, the current-limiting resistor, and the power input can be modified for different types of cell culture containers (e.g., a 6-well or 24-well plate). A 24 h of light illumination at a power of 0.2 mW/cm2 does not induce detect...

Growth factors are involved in a wide spectrum of cell functions, including proliferation, differentiation, migration, and apoptosis, and play pivotal roles in many biological events, including embryonic development, aging, and regulation of mental status1,2,3,4,5. Many growth factors signal through complex intracellular signaling cascades. These signaling events are often operated by reversible protein phosphorylation in a precisely regulated fashion6,7. Thus, an understanding of the signaling outcomes of protein kinases, which are responsible for protein phosphorylation, is fundamentally important.
Different growth factors act through a rather common intracellular signaling network, even though they stimulate distinct cellular responses8,9. Common intracellular mediators of receptor tyrosine kinases include Ras, Raf, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), phosphoinositide 3-kinase (PI3K), Akt, and phospholipase C gamma (PLC&#947;)10,11. Accumulating evidence suggests that signaling diversity and specificity depend upon the spatial and temporal regulation of signaling activity12. For instance, in rat pheochromocytoma cells (PC12), epidermal growth factor (EGF) stimulation, which results in cell proliferation, transiently activates the ERK pathway9. On the other hand, stimulation with nerve growth factor (NGF), which leads to cell differentiation, activates the ERK pathway in a sustained manner9,13. In cultured rat hippocampal neurons, transient signaling by brain-derived neurotrophic factor (BDNF) promotes primary neurite outgrowth, while sustained signaling leads to increased neurite branching14. During early embryonic development, phosphorylated ERK activity is temporally dynamic and is widespread across the embryo6. A recent genetic screen during early Xenopus embryogenesis showed that ERK and Akt signaling cascades, two downstream primary growth factor pathways, display stage-specific activation profiles7. Thus, an understanding of kinase signaling outcomes calls for tools that can probe the spatial and temporal features of kinase activity with sufficient resolution.
Conventional experimental approaches to probe the dynamic nature of signal transduction during development lack the desirable spatial and temporal resolution. For instance, pharmacological approaches utilize small chemical or biological molecules to stimulate or suppress signal transduction in cells and tissues. The diffusive nature of these small molecules makes it challenging to restrict their action to a specific region of interest15. Genetic approaches (e.g., transgenesis, the Cre-Lox system, or mutagenesis) often lead to the irreversible activation or repression of the target gene expression or protein activity16,17,18. The Tet-On/Tet-Off system19 offers improved temporal control of gene transcription but lacks strict spatial control because it relies on the diffusion of tetracycline. Recent developments in chemically induced protein dimerization20 or photo-uncaging21,22,23,24 have greatly enhanced the temporal control of signaling networks. The spatial control, however, remains challenging due to the diffusive nature of the caged chemicals.
Recent emerging optogenetic approaches, which harness the power of light to control protein-protein interactions, allow for the modulation of signaling pathways with high spatiotemporal precision as well as reversibility. Shortly after its initial success in controlling neuronal firing25,26,27, optogenetics has been extended to control other cellular processes, such as gene transcription, translation, cell migration, differentiation, and apoptosis28,29,30,31,32,33,34. A strategy using the photoactivatable protein pair Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN) was recently developed to control Raf1 kinase activity in mammalian cells and Xenopus embryos35. CRY2 binds to CIBN upon blue-light stimulation, and the CRY2/CIBN protein complex dissociates spontaneously in the dark34. Blue light excites the CRY2 cofactor, flavin adenine dinucleotide (FAD), which leads to a conformational change in CRY2 and its subsequent binding to CIBN. Constitutively active (W374A) and flavin-deficient (D387A) mutants of CRY2 can be produced through mutations in the FAD-binding pocket: the CRY2W374A mutant binds to CIBN independent of light, whereas the CRY2D387A mutant does not bind to CIBN under blue-light stimulation36,37. The optogenetic system described in this protocol uses wild-type CRY2 and CIBN to induce protein translocation-mediated Raf1 activation in live cells. It is known that the membrane recruitment of Raf1 enhances its activity38. In this system, a tandem CIBN module is anchored to the plasma membrane and CRY2-mCherry is fused to the N-terminal of Raf135. In the absence of blue light, CRY2-mCherry-Raf1 stays in the cytoplasm, and Raf1 is inactive. Blue-light stimulation induces CRY2-CIBN binding and recruits Raf1 to the plasma membrane, where Raf1 is activated. Raf activation stimulates a Raf/MEK/ERK signaling cascade. Both CRY2- and CIBN- fusion proteins are encoded in a bicistronic genetic system. This strategy can be generalized to control other kinases, such as Akt, whose activation state can also be turned on by protein translocation in cells39. This work presents detailed protocols for implementing this optogenetic strategy in mammalian cell cultures and multicellular organisms.

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light-mediated reversible modulation of the mitogen-activated protein kinase pathway during cell differentiation and xenopus embryonic development

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.

Ratiometric expression of photoactivatable protein pairs: Figure 1A shows the design of a bicistronic optogenetic construct, CRY2-mCherry-Raf1-P2A-CIBN-CIBN-GFP-CaaX (referred to as CRY2-2A-2CIBN), based on the porcine teschovirum-1 2A (P2A) peptide, which shows the highest ribosome-skipping efficiency among mammalian cell lines42. In previous work, it has been determined that the optimal ratio for CIBN-GFP-CaaX:CRY2-mCherry-Raf1 is 2:...

Watch this Scientific Journal Video about Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development at JoVE.com

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

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

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

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

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