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.
