キイロタマホコリカビ急性の機械的刺激に対する応答の評価

The overall goal of this procedure is to examine the activity of various components of the intracellular signal transduction network following brief mechanical perturbation. This method can help answer key questions in the field of cell migration such as how cells sense and directionally migrate toward diverse environmental cues including mechanical stimuli. The main advantage of this technique is that it allows examination of the initial response to mechanical stimulus without the confounding contribution of motility or polarity to the response.

We first had the idea for this method when we tested the response of adherent cells to chemoattractant which was delivered with a slight agitation and we noticed a robust response of cells stimulated with vehicle control. To begin, prepare Klebsiella aerogenes by inoculating a small number of cells from frozen stock into HL5 medium without antibiotics and incubate the culture overnight. Growth of Dictyostelium in the presence of Klebsiella aerogenes bacteria will reduce the number of macropinosomes which will improve cell responsiveness to external stimuli.

Time the experiment so that when the bacteria are ready, there are Dictyostelium growing exponentially. Collect these Dictyostelium cells and count them using a hemocytometer. Then, spread 260 microliters of the bacterial suspension containing 100, 000 Dictyostelium cells onto an SM plate.

Also for insurance, prepare plates with twice as many and half as many Dictyostelium cells. After one day of growth at room temperature, invert the plate and continue culturing the plate until the Dictyostelium cells start clearing the bacterial lawn but haven't yet begun aggregating. Then, add five milliliters of DB buffer to the plate and scrape off the Dictyostelium using a glass spreader.

Transfer the cells and solution to a 50 milliliter polypropylene tube, then rinse the plate with five more milliliters of DB and also add this to the 50 milliliter tube. Repeat if necessary. To wash the Dictyostelium, top the tube up to 50 milliliters with DB, pellet the cells, and then aspirate and discard the supernatant.

Wash the cells repeatedly until the supernatant is clear. Finally, resuspend the Dictyostelium in DB buffer to approximately five million cells per milliliter. To begin, plate two million aggregation-competent Dictyostelium onto 35 millimeter dishes with two milliliters of DB.Prepare one plate for each time point and a nonstimulated control.

At room temperature, allow the cells 10 minutes to attach to the plate. Then, wash the unattached cells off the plate using two rinses with one milliliter of DB.Once the plates are cleared of unattached Dictyostelium, incubate the attached cells in basolation buffer for 30 minutes without disturbing the plates. It is very important to avoid any movement of the plates during the half-hour incubation to prevent premature stimulation of the cells.

Now, to apply a controlled amount of mechanical stimulation, individually transfer a plate to an orbital shaker and run the shaker at 150 rpm for five seconds. Then, after the desired time, aspirate the buffer and promptly put the plate on ice. Immediately add 100 microliters of sample buffer with protease and phosphatase inhibitors and scrape off the cells.

Collect the lysate into 1.5 milliliter tubes and promptly heat them at 95 degrees Celsius for 10 minutes. Proceed with analyzing the lysate or transfer them to minus 20 degrees Celsius for storage. After making suspensions of vegetative or aggregation-competent cells, load about 600 microliters into the microfluidic chamber slide.

The inlets must be completely filled so top them off with buffer if needed. Allow the cells to attach for 10 minutes. Connect an input line for the device to an electronic pump with a 15 milliliter reservoir.

Using software control for the pump, close the valve and then fill the reservoir with DB.Now, set the pressure to 50 millibar and turn on the pump. Fill and rinse the line with DB by clicking on the valve to open it. After rinsing for about 30 seconds, turn the pressure back to zero and then close the valve.

Next, connect the line to one of the three inlets on one side of the slide. Do not trap any air bubbles and once the connection is made, cover the other two inlets on that side of the slide. Now, complete the setup by connecting the line from the drain to the single outlet on the opposite side of the slide.

Then, under a brightfield or phase illumination on a fluorescent microscope, view the channel with a 20X air objective and rinse the channel. Next, set the pressure to about 50 millibar and open the valve. Once the liquid comes out of the drain, reduce the external pressure to zero, wait about 30 seconds, and close the valve.

Next, switch to the 40X oil objective, focus on the widest part of the channel, and prepare the software to collect images every three seconds with RFP or GFP illumination. With the valve closed, set the pressure to the desired value. Then, acquire five pre-stimulation images on three-second intervals.

Next, briefly open the valve the deliver the stimulus. Now, collect at least 15 more frames of data over 45 seconds. Quantify the response as described in the text protocol.

First, load a microfluidic slide with Dictyostelium as described in the previous section. For this experiment, prepare two input lines. To close the lines, use a physical clamp on the line carrying the treatment solution and the software-controlled valve to close the line carrying the DB solution with vehicle.

Now, fill the two lines with the appropriate solution, buffer plus vehicle alone or buffer with the treatment such as an inhibitor. After the lines have been rinsed, connect them to two inlets on the side of the slide with the channel and cap the unused inlet. Then, connect the drain line to the outlet on the opposite side.

Now, wash the slide using the buffer plus vehicle as previously described. Then, fill the channel with the treatment. First, reduce the flow to zero millibars.

Next, switch the valves, flow the treatment for about 15 seconds, and start timing the incubation. Every 10 minutes or so, flow fresh treatment solution over the cells for about 15 seconds to replenish oxygen levels in the chamber. Adherent aggregation-competent cells were exposed to five seconds of shear flow using an orbital shaker.

The Dictyostelium were pre-treated with caffeine to reduce their basal activity. As a result, there was very low basal activity of PKBR1 and ERK2 and robust increase in phosphorylation of the key residues of these kinases with the stimulation. In aggregation-competent cells expressing various fluorescently-labeled biosensors, two leading edge markers, PHcrac and LimE, which are recruited by PIP3 or newly polymerized actin both showed mostly cytosolic localization in resting cells.

Following five seconds of shear flow, these biosensors rapidly re-localized to the cortex and returned to the cytosol. Effects of actin-depolymerizing drug Latrunculin A were tested using a similar shear force experiment. In this case, vegetative cells were first exposed to control conditions and then switched to the buffer that contained the desired test agent.

Latrunculin A treatment blocked the re-localization of the leading edge markers to the cortex. Intriguingly, if the flow was not shut off after two seconds but remained on for the duration of the experiment, a transient global response was still observed. Following the global response, vegetative cells migrated against the flow.

Proper preparation of cells is extremely important for the success of these assays. Vegetative cells have to be grown in association with bacteria to ensure their responsiveness to mechanical stimulation. For aggregation-competent cells, cells that are either under or over developed, tend to respond poorly.

Biochemical and microscopic approaches compliment each other and that's allowed us to assess a large number of signal transduction intermediates for their response to mechanical stimulation. By adding pharmacological as well as genetic perturbations, we can learn how mechanical stimuli are perceived and transmitted. This is important for understanding directed cell migration that is guided by physical stimuli such as shear flow.

Since mechanical stimulation triggers activation of the same signal transduction network as chemoattractants, we can now examine the integration of various stimuli on cells. Ultimately, these studies will help us understand how migrating cells such as our own immune cells and metastatic cancer cells navigate through the body.