Experiment 8: Microtubules and cell motility
The purpose of this experiment is to determine the effect of a drug (nocodazole) that binds to free tubulin and prevents microtubule polymerization on microtubule dynamics and cell behavior. We will start by testing different concentrations that have been reported in the literature as effective on You will test different concentrations of the drug to determine how time of treatment vs. amount of drug affects motility. Then, you will wash out the drug to determine how rapidly cells recover. On Thursday, we will do the same, but this time directly image the effect of the drug on microtubules. It would be nice to do both at the same time, but the microtubules are so 3-dimensional, that it is very difficult without a confocal microscope. Instead we will test motility effects, and then later flatten the cells under agarose to make them more two-dimensional (but less motile) and image the effects of the drug on fluorescent microtubules.
You will be given cells that were transfected with a vector that drives expression of GFP-tubulin which makes microtubules fluorescent. You can do the first part of the experiment on wild-type cells (or your GFP-filabd cells).
Nocadazole
- Binds beta-tubulin, preventing tubulin polymerization.
- We have stock solutions of 3mM in DMSO, 0.3 mM in DMSO and 0.03mM in MCPB for you to use. FW is 301g/mole.
- The solvent DMSO can have its own effects on cells! So, when you use this drug, you need to make sure the DMSO is diluted at the least 1:100 dilution (1% final) to minimize its effects on cells. Also, when making dilutions from DMSO, mix well, as it is more dense than water. For the same reason, do not add DMSO or drug in DMSO directly to dishes of cells. It will rapidly sink to the bottom and expose your cells to very high concentrations of drug and DMSO.
These drugs are toxic (just look at what they do)! Wear gloves when handling solutions containing high concentrations of these drugs. The amounts we are working with and the concentrations are very low and generally not of great concern.
Part I: Effect of Nocodazole on Motility
1. Plate cells in P30 dishes. If you are just doing transmitted light, you can use HL5. If you plan to use fluorescent cells and image them, then use MCPB or LoFlo.
2. Wait 30-60 minutes for them to attach and begin moving.
3. Image for long enough to establish motility rate
4. Add Nocodazole to cells and continue imaging
1.Drug concentration: The target concentration for microtubule inhibition in the literature is about 10-30 µM. You should not need to go higher than this. Try lower concentrations as well to see if there is an effect. I would suggest going at least 3-5x lower so you cover a wider range.
b.The easiest way to treat the cells while imaging is to plate your cells and allow them to attach and then remove some media before you start imaging. Then add back that media iwth drug to bring them to the target concentration. For instance, if you plan to use 1 ml of media in a P30 dish, plate the cells in 1ml, allow them to attach and then remove 250µl of media. Add the amount of drug needed for 1 ml total to the 250 µl, mix and add that back to the dish when you want to start the drug treatment. Adding a fairly large volume of media means you will get good mixing of the drug as you add it. If you added 5µl of drug to 1 ml, it would sink to the bottom and take a long time to equilibrate because you can’t mix while imaging.
5.Presuming there is an effect on motility, wait until it stabilizes, then gently wash out the drug and measure the time it takes the cells to recover.
Part II- Imaging the effect of nocodazole on microtubules
We will provide you with cells expressing GFP-tubulin
In order to image microtubules, we are going to try an under agarose imaging technique. The idea is to flatten the cells under a layer of agarose so microtubules are all in focus in the 2D volume of the flattened cells.
1.Plate cells in an iBidi dish ( we will give each group 2 dishes so that you can ready one while imaging the second. Use a high concentration of cells so that there will be many cells in each field of view of the 100x oil objective. Allow them to attach and then change the media with MCPB containing ascorbic acid. Allow them to attach for 30-60 minutes.
2.Meanwhile, prepare the agarose overlay. Melt 0.5g of agarose in 25 ml of MCPB in the microwave. Allow to cool somewhat and then add ascorbic acid.
3.While it is cooling, prepare glass slides for making the overlay. Get two glass slides and 4 #1.5 18x18 mm coverslips. Position the coverslips with two stacked at each end of the slide.
4. Add about 1 ml of the melted agarose solution with a 1ml pipette spread out over the central area of the slide and then quickly put the second slide on top. You are essentially using the coverslips as a spacer to create a 340µm thick sheet of agarose.
5. Allow the agarose to harden for 5-10 minutes and then peel the slides apart. The agarose will stick to one slide.
6. Use a #10 cork borer to cut a round agarose sheet smaller than the size of the inside of the iBidi dish. Alternatively, you can cut small squares of agarose with a razor blade. You can make many sheets from one slide. After you cut your first overlay sheet, place the rest of the sheet in a petri dish with buffer + ascorbic acid if it is going to sit for a while so it doesn’t dry out.
7.When you are ready to overlay the cells, add the agarose sheet to the iBidi dish with cells and allow it to float down (or gently push it) onto the cells. Now begin removing the media with your P1000 pipettman. You want to remove as much media as you can.
One of the keys is to not slide the agarose sheet around while it is compressing the cells. That will eventually kill the cells.
8.Now use strips of absorbent paper to gently blot the rest of the media off the top of the agarose and then from underneath by blotting the edges of the agarose (where it meets the plastic) so you are removing the liquid from underneath the agarose. The more you blot, the more flattened the cells will become. You can see this change in phase contrast as the cells begin to look like round pancakes as they are flattened.
9.When you have them flattened, pipette some MCPB or water onto the lip around the edge of the dish to provide some humidity so the agarose does not dry too much. You can leave the lid on while you are setting up to prevent drying, but put it on upside down to make it easier to remove later.
10.Set up the dish on your microscope with the 100x oil objective and get set up to do phase contrast transmitted light plus blue illumination fluorescence (for GFP-tubulin).
11.Take the lid off the dish and begin to Image the cells at about 10 second intervals.
12.When you done enough to have a good sense of microtubule behavior, switch to a new field , remove the lid and add the nocodazole in a small volume (about 5 µl) to the top of the agarose sheet. The drug will slowly diffuse throughout the agarose and eventually reach the cells.
a. To calculate the concentration of drug to use, determine the volume of the agarose sheet and calculate the amount of nocodazole to put in 5 µl to bring the concentration of the entire sheet to the desired amount.
13. Continue imaging and if you start with the higher concentration of nocodazole you should see the disappearance of the microtubules
14.When complete, you can try reversing the drug effect. This is challenging but should work. Add about 0.5 ml of MCPB to the center of the dish and let it sit for a few minutes to diffuse through the agarose (roughly the time it took to see an effect on the cells and then some).
15.Gently remove the buffer and repeat once (you should not have to wait long the second time- you are just trying to get rid of excess drug).
16.Remove the MCPB and blot the agarose sheet dry as you did in the beginning to reflatten the cells.
17.Put them back on the microscope stage and start a time lapse to see how long it takes the microtubules to regrow.
Key questions to think about:
1. What is the relationship of inhibitor concentration to the rate and extent of microtubule depolymerization or motility? You can quantify this either by the length of the microtubules, or possibly by the increase in cytoplasmic fluorescence as more of the tubulin becomes cytoplasmic. A key question for you to address is the relationship between concentration and timing of effect. Does more Nocodazole lead to faster depolymerization or more extensive depolymerization or both? If you use less, does the same thing happen, but take longer or is depolymerization not as extensive.
2. Does inhibition or recovery of motility and microtubule length happen on the same time scale? What might it mean if it takes much longer for one effect than the other?
Media and Reagents :
MCPB
1.42 g Na2HPO4
1.36 g KH2PO4
0.19 g MgCl2
0.03 g CaCl2
0.5 Streptomycin dihydrosulfate
Fill up to 1L with H20
Adjust pH to 6.5-6.7
Filter-sterilize
Cell strains:
NC4A2-GFP-FilABD
NC4A2-GFP-tubulin
LoFlo
1ml 1000x FM salts 1
1ml 1000x FM salts 2
0.1 ml 10000 FM trace elements
11 glucose/1H2O
5ml 1M K2HPO4
5g casein peptone
ddH2O to 1000ml, pH to 6.5
filter sterilize
add amp/strep
Nocodazole - 0.03, 0.3 mM prepared from stock of 3 mM in DMSO
Other equipment and supplies needed for this experiment:
1.8-well chamber slides or 30mm iBidi dishes
2.centrifuge
3.15-ml falcon tubes
