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 ...

The overall goal of this experiment is to use light to control the mitogen-activated protein kinase signaling pathway in intact cells and in Xenopus embryos. This method can help answer key questions in cellular and developmental biology, such as how temporal modulations of kinase activity regulates self-determination during cell differentiation, and embryonic development. The main advantage of this technique is that the mitogen-activated protein kinase activity can be controlled reversibly in intact cells and in multicellular organisms, such as developing embryos.This method can provide insight into signaling kinetics during Xenopus embryonic developments. It can also be applied to other model systems such as socotra, zebrafish, or mouse. Today, a graduate student from my laboratory, Vishnu Krishnamurthy, will demonstrate the procedure.And Aurora Turgeon, a post-star in my laboratory, will demonstrate all procedures involved in Xenopus embryos. First, assemble the cell culture device by placing one autoclaved sterile PDMS chamber on a sterile PLL-coated cover slip in a 60 mm petri dish. Next, use 0.5 mL of 0.25%trypsin to detach pHK21 cells from one well of a 6-well tissue culture plate.After counting the cell density with the hemocytometer, seed the chamber with 20, 000 cells in 200 microliters of cell culture medium. 24 hours after cell plating, transfect the cells with 50 to 100 nanograms of CRY2-mCherry-Raf1 2A2CIBMG of PCaA X plasmid as per the manufacturer's protocol. Three hours after transfection, change the medium to 200 microliters of fresh culture medium and allow the cells to recover overnight By incubating the culture in a 37 degree Celsius and five percent carbon dioxide incubator.The next day, begin optogenetic induction and imaging by first replacing the cell culture medium with 200 microliters of carbon dioxide independent medium. Then set up the data acquisition protocol. Select 488 nonometer excitation FITC channel for optogenetic stimulation, and the 561 nanometer excitation CRITC channel to track the cellular localization of mCherry labeled protein.Measure the power of the blue light by placing a power meter close to the objective window. A total power of two microwatts is sufficient to induce CIBN CRY2-PHR association. Set up a time stamp acquisition with the five second interval.And the total acquisition time of two minutes. Apply appropriate index-matching material on the objective window. Place the cell chamber on the microscope stage and use the bright field mode to focus on the cells on the coverslip surface using the eye pieces.Next, move the microscope stage to locate a transfected cell under green light. Record a series of time stamped images in both the FITC and CITC channels. The basic sample image shows CIBNGPCAAX localized on the plasma membrane.This is a snapshot of CRY2 mCherry raf1 before blue light simulation, whereas this images shows a snapshot of CRY2 mCherry raf one after ten pulses of blue light stimulation. Make the LED array by inserting tonal blue LEDs into two breadboards and connecting with current limiting resistors. Place the breadboards into an aluminum box.Insert two metal wires to connect the boards to the power supply. Make sure that the length of the wire is sufficient when the light box is placed inside a carbon dioxide incubator. Then place a transparent light diffuser to act as the cover of the light box.Lastly, calibrate the power output of each LED at a range of voltage inputs. Use a power of zero point two milliwatts per square centimeter for the 24 hour PC12 cell differentiation assay. Place the LED array in a 37 degree Celsius incubator supplemented with five percent carbon dioxide.Connect it to the power supply using the metal wires and set the power of the LED to zero point two milliwatts per square centimeter. Place the 12 well plate containing transfected PC12 cells on the window of the LED array. Align the plate so that every well is fully illuminated.Apply continuous light illumination for 24 hours. To image the differentiated cells, set up single snapshot data acquisition. Use 200 milliseconds for both the GFP and TexasRed channels.Capture images of the transfected cells in both the GFP and TexasRed channels, and save the files for data analysis. Begin this procedure by fabricating needles for RNA microinjection by pulling glass capillaries with a capillary puller. Remove the jelly coat from the embryos by treating them with three percent cysteine diluted in 0.2 XMMR for 15 minutes.Transfer the embryos to three percent polysucrose And zero point five XMMR solution for microinjection. Accurately inject 500 picograms to one nanogram of CRY2-mCherry-Raf1-2A-2CIBN-GFP-CaaX RNA into each embryo. Then culture the microinjected embryos in the microinjection solution at room temperature.When the embryos reach the med gastrula stage, transfer them to 0.2x MMR solution, and continue to culture until the desired stage of development. Then place the 12-well plate on the home built LED array for blue light treatment. Place a mirror on top of the 12-well plate to ensure full blue light illumination of the embryos.Harvest the embryos for histological, western blot, or gene expression analysis after exposure to blue light for the desired time. This image shows multichannel snapshots of PC12 cells tranfected wth CRY2-2A-2CIBN after 24 hours of blue light stimulation at zero point two milliwatts per square centimeter. Both the GFP and mCherry reporters are seen.Circles mark differentiated cells and squares mark undifferentiated cells. Here the cells were treated the same way as those in the prior image, except no blue light stimulation was used. No differentiation is seen.These cells show representative results after transfection with raf1 GFPCaaX, a constitutively active raf1. The circles and rectangles mark differentiated and undifferentiated cells respectively. This histogram shows differentiation ratios of PC12 cells transfected with CA-Raf1, Co-transfected with CRY2 mCherry raf1 and CIBN GFP Caax and singly transfected with CRY2A 2CIBN.Only cells exposed to blue light showed significant differentiation. This image shows the morphology of normal Xenopus embryos without MRNA injection, and without light treatments. This image shows embryos injected with CRY2 2A2CIBN MRNA and subjected to blue light stimulation.Activation of raf1 by treating CRY2 2A2CIBN injected embryos with blue light induces ectopic tail-like structures in the head region. Following this procedure, one can answer additional questions, such as how stage specific activation of the mitogen activated protein kinase pathway regulates gene expression. This optogenetic method can be generalized to control other signaling pathways with similar activation mechanisms.

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