This method can help answer key questions in the fields of cell and developmental biology, such as which proteins are involved in the regulation of non-muscle myosin-II contractility and which proteins are key in the folded gastrulation or fog-signaling pathway. The main advantage of this technique is that, coupled with RNEI depletion, the cellular-contractility assay can be used as a tool for gene discovery for proteins involved in fog signaling and non-muscle myosin-II contractility. The implications of this technique extend toward our basic understanding of development.
Non-muscle myosin-II contractility is universally critical to cell-shape change, or morphogenesis, that occurs during development. Though this method can provide insight into the development of drosophila, it can also be applied to other systems, because it is critical to a whole host of other cellular functions. Generally, individuals new to this method will struggle because getting correct cell density and recognizing the difference between contracted and non-contracted cells can be difficult.
To begin, coat the glass portion of 35-millimeter glass-bottomed dishes with 200 microliters of con-A solution. Incubate the dishes for approximately two minutes at room temperature in a tissue-culture hood. After this, remove the con-A solution, and allow the dishes to air-dry completely.
Add approximately two milliliters of fresh cell-culture media to each of the glass-bottomed dishes. Then, resuspend the S2R+cells by pipetting the cell-culture medium up and down. Transfer the resuspended S2R+cells to the prepared glass-bottomed dishes.
Then, check the cell density under a tissue-culture microscope by focusing up and down through multiple planes of cells. Establishing the initial cell density is critical. Too few cells will make it difficult to quantify, and too many cells will make quantification tedious.
Use the microscope to focus up and down the dish to estimate the number of cells. Next, allow the cells to attach to the con-A-coated glass-bottomed dishes for 45 to 60 minutes. First, gently pipette to remove all of the cell-culture medium from the attached S2R+cells.
Then, carefully add back 75 microliters of fresh medium. Next, add 75 microliters of fog-conditioned media to the glass portion of the dish. Using phase-contrast microscopy at 20 times or 40 times magnification, monitor the contractility of an entire field of cells.
Track the morphology of the cells for the alternating pattern of phase dark-and-light ruffling, characteristic of shape change. Correctly identifying cells undergoing contraction can be hard the first time. As the cells contract, phase dark and light ruffles will form, making the cell appear like a starburst.
Fix the S2R+cells with a 10%paraformaldehyde solution. Then, pipette to remove the cell-culture medium from the fog-treated S2R+cells, and replace it with 1.5 to two milliliters of the paraformaldehyde solution. Incubate both the S2R+cells and the fog-treated S2R+cells for 15 minutes at room temperature.
Next, use a pipette to remove the paraformaldehyde solution, and dispose of the waste properly. Then, rinse the cells with PBS three times to remove any residual fixation solution. Block the cells with 200 microliters of 5%normal goat serum in PBS supplemented with 0.1%PBST for 20 minutes at room temperature.
Next, dilute the primary antibody in PBST, using a volume of approximately 200 microliters to completely cover the glass portion of the dish. Use a pipette to remove the blocking solution, and add the primary antibody solution directly to the glass portion of the dish. Then, incubate for one hour at room temperature.
After this, use a pipette to remove the primary antibody solution, and rinse the cells with fresh PBS three times. To prepare the secondary antibody solution, dilute the secondary antibody in PBST. Then, add the secondary antibody solution directly to the glass portion of the dish.
Incubate the dish for one hour at room temperature. After this, use a pipette to remove the secondary antibody solution, and rinse the cells with fresh PBS three times. Then, add sufficient anti-fade fluorescent mounting medium to cover the glass portion of the dish.
Store the samples at four degrees Celsius away from any light. First, use 20 times or 40 times magnification to capture 10 to 30 non-overlapping image fields of the fixed cells. Finally, use a standard cell counter to count and record the number of contracted and non-contracted cells in each of the captured fields.
Using this protocol, S2 and S2 fog-myc cells were grown and treated with copper sulfate to induce the expression of fog-myc. Unsurprisingly, fog-myc was not detected in the S2 cells under any conditions. However, fog was detected in media harvested from the S2 fog-myc-stable cell line as early as 24 hours after induction with copper sulfate.
Approximately five minutes after treatment with fog-myc, the RLC formed rings indicative of non-muscle myosin-II constriction. In a representative cellular-contractility assay, the phase-dense ruffles indicative of constriction were prominent in control-depleted cells treated with fog-conditioned media. Rho-depleted cells treated with S2-conditioned medium demonstrated typical smooth-edged fried-egg-like morphology.
While attempting this procedure, it's important to remember time and the quality fixation is important. After 10 minutes, cells begin to relax, so the cells should be fixed before this time. Poor fixation conditions can lead to the false positives.
Using this assay, researchers identified mist, one of the two fog coreceptors. This assay was also used to get a better understanding of the role of rigate during fog signaling.
