The overall goal of this procedure is to apply advanced fluorescence confocal microscopy to monitor spatiotemporal dynamics of signaling events during chemo sensing in dictyostelium discoid dium cells first chemotactic competent dictyostelium discoid dium cells are prepared. Next, a controlled chemo attractant gradient is imaged to establish a linear relationship between the chemo attractant concentration and the fluorescent intensity chemo attractant. The third step is to obtain an immobile cell system for imaging signaling events involved in cyclic A MP gradient sensing.
The final step is to simultaneously monitor two G PCR R controlled signaling events in live cells, heterotrimeric G-protein activation and PIP three production. Ultimately spatiotemporal changes in fluorescence intensities in single cells that are simulated with a chemo attractant. Cyclic A MP can be visualized through live cell imaging with advanced confocal microscopy.
The main advantage of this technique is to allow us to obtain spatial temporal information on signaling event in life cells. This lifestyle measurements permit a better understanding into how A-G-P-C-R signaling network is able to detect a chemo attractant ingredient and to generate proper responses. Demonstrating the procedure will be Dr.Shu, who is an expert in lifestyle imaging methods To generate dsco dium cells that are chemotactic to the chemo attractant cyclic A MP harvest cells growing in D three trich media from a shaking culture at 22 degrees Celsius.
Wash cells twice in non nutrient developmental buffer by centrifugation at 400 Gs for five minutes. At room temperature, resus suspend the cells in the DB buffer at a density of two times 10 of the seventh cells per milliliter. Transfer 20 million cells in 10 milliliters of developmental buffer to a 250 milliliter flask, and then shake the cells at 100 RP M at 22 degrees Celsius for one hour.
Then deliver 100 microliters of 7.5 micromolar cyclic a MP stock to the 10 milliliters of cells every six minutes over the next six hours to achieve a final concentration of 75 ano molar cyclic A MP.Now collect the cyclic A MP chemo attractant competent cells by centrifugation this time at 200 Gs, and then re suspend the cells with DB buffer containing 2.5 millimolar caffeine. Shake the cells again at 22 degrees Celsius for 20 minutes. This time at 200 RPM to salate the cells for chemotaxis.
First backfill a micro pipette with a freshly prepared 30 milliliter solution of one micromolar cyclic A MP and Alexa 5 94 at 0.1 micrograms per microliter in DB buffer. Then attach a Femto tip to a micropipet holder and connect the tubing to a pressure supply apparatus. To establish a stable gradient, attach the micro pipette assembly to a motorized micro manipulator.
Fill a one well lab tech chamber with six milliliters of DB buffer and then mount the chamber over a 40 x oil lens on a confocal microscope using Brightfield optics center, the Femto tip in the field of view. Next, turn on the pressure supply and set the compensation pressure to 70 Hector Pascals to establish a gradient of the cyclic A MP Alexa 5 94 mixture. Visualize the cyclic A MP gradient by monitoring the mixture of the desired concentration of cyclic A MP and Alexa 5 94 fluorescence using an excitation with a 543 nanometer laser line.
Finally, use the auto positioning function of the micro manipulator to put the micro pipette to the desired positions and set position one, position two, and position three in order to manipulate the gradient to which cells are exposed. After caffeine treatment, remove an aliquot of cells and pellet the cells at 500 Gs for three minutes. At room temperature, remove the buffer and dilute the cells to five times 10 of the fifth cells per milliliter.
With fresh DB buffer containing 2.5 millimolar caffeine. Then apply one milliliter of the cell suspension to a single well chamber. After allowing the cells to adhere for 10 minutes, carefully pipette off the buffer to remove unattached cells and then replace with the same volume of fresh DB buffer with caffeine to start imaging, locate the desired cells under the microscope.
Then to monitor the dynamics of the signaling components in cells exposed to a steady gradient. Treat the cells with five micromolar LA truculent B for 10 minutes prior to any experiments. This scheme shows a brief signaling pathway of directional sensing in this movie.
It can be seen how a cyclic A MP gradient induces rapid chemotaxis of D discost cells, cells expressing the PIP three probe. P-H-G-F-P appear in green. The gradient as visualized by Alexa 5 94 appears in red.Here.
A simple method to obtain a linear relationship between cyclic A MP concentration and the intensity of a fluorescent dye. Alexa 5 94 by a dilution series of two micromolar cyclic A MP mixed with 10 micrograms per milliliter of Alexa. 5 94 is shown.
Next, an easy way to establish a quantitative measurement of the cyclic A MP concentration of a gradient by the intensity of Alexa 5 94 is graphically demonstrated. This figure shows how cells that have had their motility suppressed by lat truculent B an acton polymerization inhibitor still maintain their directional sensing capability. The cells express the PIP three probe, P-H-G-F-P and appear green while the gradient in red is visualized by Lexi 5 94.
In this movie, it can be seen how the employment of a non-polarized simplified cell system and manipulatable cyclic A MP stimulation can address fundamental questions of directional sensing. To demonstrate the application of live cell imaging with a high tempos spatial resolution, here, a cell in green exhibiting a biphasic expression of PIP three. In response to exposure to a steady cyclic A MP gradient in red is shown.
This image shows the regions of interest for the measurement of the kinetics of PIP three production. The kinetics of PIP three production in the front and back of the cells upon exposure to a steady gradient is presented here with PIP three production on the front and back, represented in black and gray respectively. Applying live cell imaging, we have systematically measured the dynamics of a directional sensing specific signaling network from cyclic A MP stimulation to PIP three production.
This scheme shows the signaling network of directional sensing from cyclic A MP stimulation to PIP three production. The kinetics of each component is presented with a line in the same color. In this first figure, how a uniformly applied cyclic A MP stimulation triggers a persistent G protein activation, which triggers a transient PIP three production is represented in this movie.
A rainbow of images of G and pH cells shows that a uniformly applied cyclic A MP stimulation triggers a persistent G-protein activation at the cell periphery, which simultaneously triggers a transient PIP three production. Here, the kinetics of G protein activation and PIP three production upon a uniformly applied cyclic A MP stimulation are shown. While trying these experiments, it is important to keep in mind that the key is to obtain reactive cells, different genetic background of the cells and environmental conditions.
Imaging acquisition conditions all changes the cell physiology and may make the cell less responsive. Following this procedure, computational modeling can be applied to answer additional questions such as how dynamic interactions within a signaling network give rise to a cell behavior. This methods pave the way for researchers in the field of eukaryotic chemo taxes.
We can develop a computational model of chemoattractant sensing. This model can be used to simulate the dynamics of a signaling event. Interplay between model refinement and the lifestyle measurement can lead to a deeper understanding of chemo taxes.
After watching this video, we hope that you have a better understanding of how to apply advanced fluorescence confocal microscopy to monitor signaling event in life cells. Hopefully you can apply this method to your study.
