Gerando um "humanizado"

The overall goal of this experiment is to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of Kinesin-5 motor, so they can be used to study error correction. Error correction during cell division is a process in which erroneous kinetochore and microtubule interactions are destablilized, to give the cell a second chance, or another opportunity, to form proper attachments so then the genome can be properly segregated. This method can help answer key questions in the cell biology field, related to error correction process.

The main advantage of this technique is that we can use a Drosophila S2 cell line that have fewer chromosomes, and are sensitive to a Kinesin-5 inhibitor, both in which facilitates the visualization of individual error correction events. Beginning with a previously cultured 25 centimeter square flask of 100%confluent GFP-Alpha Tubulin expressing S2 cells, transfer two milliliters of the culture to a 35 millimeter dish. Then, incubate the cells at room temperature for approximately one hour to semi-adhere.

Remove the medium from the dish, and add one milliliter of Schneider's medium, supplemented with FBS, into two micrograms of the Eg5-mCherry construct and transfection reagent to transfect the cells following the manufacturer's protocol. Use Parafilm to seal the dish, and incubate the cells at 25 degrees Celsius for 16 to 18 hours. Then add one milliliter of Schneider's medium and return the cells to the incubator.

On day three post-transfection, transfer 500 microliters of transfected cells into a new 35 millimeter tissue culture dish containing 1.5 milliliters of Schneider's medium for a test induction. To induce expression of Eg5-mCherry, add copper sulfate to a final concentration of 500 micromolar. Seal the dish in Parafilm and incubate at 25 degrees Celsius overnight.

Place a previously prepared 22 millimeter by 22 millimeter Concanavalin A coated cover slip in a clean 35 millimeter tissue culture dish. Then seed 500 microliters of the 100%confluent induced cells onto the cover slip, and allow the cells to adhere at room temperature. To check for Eg5-mCherry expression, assemble the cover slip into a rose chamber with medium and image at room temperature using a fluorescence microscope, according to the guidelines in the text protocol.

After ensuring that the cells are expressing both Eg5-mCherry and GFP alpha tubulin, transfer the remainder of the transfected, uninduced cells to a 25 centimeter square tissue culture flask, containing four milliliters of Schneider's medium. Add Blasticidin S HCL at a final concentration of 25 micrograms per milliliter to the flask, and continue splitting the cells in the presence of Blasticidin S HCL until cell death ceases. After preparing KLP61F double-stranded RNA according to the text protocol, seed S2 cells expressing Eg5-mCherry to a 35 millimeter culture dish at 25%confluency, and allow them to adhere for one hour.

Remove the medium from the dishes and add one milliliter of serum-free Schneider's medium to five micrograms of double-stranded KLP61F RNA. After incubating at room temperature for one hour, add one milliliter of Schneider's medium with 10%FBS. Incubate the cells at 25 degrees Celsius.

24 hours after dsRNA treatment, induce expression of Eg5-mCherry by adding copper sulfate to a final concentration of 500 micromolar. Incubate the cells at 25 degrees Celsius for an additional 24 hours. To carry out live cell imaging, place a 22 millimeter by 22 millimeter Concanavalin A coated cover slip into a clean 35 millimeter tissue culture dish.

Seed 500 microliters of the dsRNA treated cells that have been induced overnight onto the cover slip, and allow them to adhere for about one hour. After collecting time-lapse images of bipolar spindles according to the text protocol, remove the medium from the rose chamber and add Schneider's medium containing one micromolar STLC and replace the STLC medium two more times, discarding the medium after each wash to visualize spindle collapse. To wash out the drug and reverse spindle collapse, carefully remove the STLC-containing medium from the rose chamber and discard it in a waste container, and use 6 to 8 millimeters total of Schneider's medium to wash the cells four times, discarding the media after each wash.

Then after adding fresh medium to refill the chamber, continue imaging. To get access to the medium for drug washing and washout, carefully remove the top cover slip from the rose chamber while secured on the microscope. Alternatively, a glass-bottom Petri dish treated with Concanavalin A similarly to the cover slips can be used for live cell imaging.

After seeding 500 microliters of S2 cells treated with KLP61F dsRNA onto a Concanavalin A cover slip and allowing the cells to adhere, add 1.5 milliliters of Schneider's medium to bring the final volume to two milliliters. Once cells have been arrested in mitosis according to the text protocol, add one micromolar STLC and incubate for one hour to allow monopoles to form. Wash out the STLC by using two milliliters of fresh Schneider's medium to rinse the cover slips three times.

To fix the cells at different time points of bipolar spindle formation, and to assess kinetochore attachment sites, begin by using two milliliters of 1X BRB80 to quickly rinse the cover slips. In a chemical hood, add two milliliters of 10%PFA in 1X BRB80, and incubate for 10 minutes. Next, to permeabilize the cells, add 1%Triton X in 1X PBS and incubate for 10 minutes.

Then use two milliliters of 0.1%Triton X in 1X PBS to wash the cells three times. Next, transfer the cover slip, cell side facing up, onto a sheet of Parafilm in a 150 millimeter Petri dish. Add 150 microliters of Boiled Donkey Serum, or BDS, to the cover slips, and incubate at room temperature for one hour to block nonspecific antibody binding.

After the incubation, remove the block and add 150 microliters of primary antibodies prepared according to the text protocol before incubating at room temperature for one hour. After washing the cover slips three times, add Fluoro-4 conjugated secondary antibodies diluted in BDS and incubate at room temperature for 30 to 60 minutes. Once the cover slips have been washed three times, incubate them with 150 microliters of BDS, containing one microgram per milliliter of DAPI for five minutes.

Then after the cover slips have been washed twice, use eight microliters of mounting medium to mount the sample, cell side down, on a three by one inch slide. Use nail polish to paint the corners. Finally, use a 100X objective to image cells with bipolar spindles expressing Eg5-mCherry.

Take a Z series, consisting of 30 planes at 0.2 micrometer intervals in all channels. Carefully analyze the kinetochore and microtubules to determine the attachment state. KLP61F is required to form bipolar spindles in Drosophila S2 cells;however, small molecule Kinesin-5 inhibitors are ineffective against it.

These images show that the human Kinesin-5 Eg5 can rescue the function of KLP61F in Drosophila S2 cells. When the Kinesin-5 inhibitor, STLC, is added, microtubule-associated levels of the motor drop, and the spindle collapses, resulting in a monopole in cells lacking endogenous KLP61F as the result of successful RNA I.This figure demonstrates that Kinesin-5 inhibition can be reversed by washing out the drug, and recovery of a bipolar spindle can be followed over time. Upon removal of the inhibitor, Eg5-mCherry reassociates with the microtubules, and the cells can progress into a bipolar anaphase.

After watching this video, you should have a good understanding of how to generate a Drosophila S2 cell line that is sensitive to a small molecule inhibitor of Kinesin-5. Once mastered, this technique can be performed in about six hours, if performed properly. While attempting this procedure, it's important to plan ahead, because this method requires preparation a few days in advance.

Don't forget that working with small molecule inhibitors can be hazardous, so always wear gloves. Building humanized fruit fly cells can be proved through the application of Crispr-Cas 9 technologies, and replacing the Drosophila Kinesin-5 by its human counterpart. Development of humanized Drosophila cells should advance our understanding of error correction and other essential cellular processes in the future.