调查Coronin A在早期饥饿反应中的作用

The overall goal of this experimental procedure, is to investigate the Coronin-A dependent early starvation response of Dictyostelium discoideum. This method can help answer key questions in the field of Developmental Biology, such as the identification of signaling pathways and factors involved in the early development of Dictyostelium discoideum. The main advantage of this technique is that it has the potential to provide a tool for automated and high-throughput screening of development inducing factors and proteins involved in early developmental signaling.

Generally, individuals new to this method, will struggle because of multiple variables inherent to the assay, such as cell densities, time of adherence, and time of development. To begin this procedure, grow DH1.10 cells or corA-deficient cells in an Erlenmeyer flask containing HL-5 medium, at 22 degrees Celsius, in a shaking incubator at 160 rpm. Keep the cells at a density between one times 10 to the four, and two times 10 to the six cells per milliliter.

To examine cell aggregation, harvest 10 to 50 milliliters of log-phase-growing DH1.10 cells or corA-deficient cells generated in the DH1.10 background. Centrifuge them for three minutes at 400 times g. Then was the cells twice in BSS.

Afterward count the cells using a hemocytometer. Subsequently, plate the cells in a 24 well-plate. Allow them to adhere for one hour at 22 degrees Celsuis in BSS.

Then, visualize the cell aggregation with time-lapse microscopy, and take images every 135 seconds. To examine the affect of the externally applied cyclic AMP pulses on the development of DH1.10 cells, or DH1.10 corA-deficient cells, harvest 10 to 50 milliliters of the cells and centrifuge them for three minutes at 400 times g. After that, wash them twice in BSS.

Count the cells using a hemocytometer. Subsequently, re-suspend the cells to a density of one times 10 to the seven cells per milliliter in BSS. Then, shake the cultures at 22 degrees Celsius for two hours at 160 rpm before applying pulses.

Next, apply cyclic AMP pulses using a timer controlled peristaltic pump. Program the pump to deliver a five second pulse of cyclic AMP every six and a half minutes over a period of five hours. A critical step is to ensure that the tip of the cyclic AMP injection is not continuously submerged in the solution, but only submerged periodically in the waves generated by the shaker.

After cyclic AMP application, count the cells again using a hemocytometer. Then, plate the cells in a 24 well-plate and allow them to adhere for one hour in BSS. After 16 hours, visualize the cell aggregation at 22 degrees Celsius, with bright-field microscopy using a five x subjective.

In this procedure, prepare fresh conditioned medium, by collecting log phase DH1.10 cells or DH1.10 corA-deficient cells from the shaking cultures with HL5 medium, at 22 degrees Celsius. Centrifuge the cells for three minutes at 400 times g. After that, wash them three times in PBM.

Subsequently, count the cells using a hemocytometer, Then, re-suspend them in PBM at a density of one times 10 to the seven cells per milliliter and shake them for 20 hours at 22 degrees Celsius at 110 rpm. Next, centrifuge the cells at 400 times g for three minutes, followed by collecting the conditioned medium. Afterward, clarify the medium by centrifugation at 8, 000 times g for 15 minutes at four degrees Celsuis.

Then, filter the conditioned medium through a 0.45 micrometer filter and dilute it three-fold in PBM. Count the cells using a hemocytometer, plate them in a 24 well-plate, and allow them to adhere for one hour at 22 degrees Celsius. Next, exchange the supernatant of the cells with a previously prepared conditioned medium.

After 16 hours, visualize the cell aggregration with bright-field microscopy using a five x subjective. Here, the cells deficient in Coronin-A show a defect in early development, and were unable to form multi-cellular aggregates, which is the initial step during the developmental cycle of Dictyostelium discoideum. In this figure, the vegetative Wild type, and corA-deficient cells, were washed and starved for two hours before being pulsed with, or without, 15 animal or cyclic AMP, every six and a half minutes for five hours in suspension.

The application of exogenous cyclic AMP pulses, fully restored the Wild type phenotype. In this figure, exponentially growing Wild type and corA-deficient cells, were washed in PBM, seeded into multi well-plates, and incubated in conditioned medium obtained from Wild type, or corA-deficient starving cells. The images were taken 16 hours after seeding the cells.

corA-deficient Dictyostelium discoideum, were able to produce all required factors for the developmental induction, but were unable to respond to them. Once mastered, this technique can be done in 20 hours, including incubation time, if it is performed properly. While attempting this procedure, it's important to remember, that for different strains an adjustment of multiple variables of the assay, such as cell densities, time of adherence, time of development, will be required.

Following this procedure, biochemical fractionation and subsequent proteomic analysis, can be performed in order to identify factors involved in the early starvation response of Dictyostelium discoideum. After watching this video, one should have a good understanding of how to induce early development in Dictyostelium discoideum under submerged conditions, using either conditioned media, as well as cyclic AMP pulses.