Cell Division

1. Observing the Cell Cycle in a Root Tip Expand
   • Hypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a chemical that interferes with microtubular polymerization, all of the cells will be arrested at the same stage of the cell cycle and that in untreated onion tip slices all of the different stages of the cell cycle will be visualized. The null hypothesis is that there will be no differences in mitosis between the two groups and that different stages of the cell cycle will be visualized.
   • To prepare for this experiment you will use an onion that has been placed in water for several days until new white root tips have begun to sprout. Use a razor blade to cut a 1 cm piece from the end of one of the root tips.
   • Place the slice of onion into a microcentrifuge tube labeled “N” for the nocodazole condition.
   • Then, cut a second 1 cm piece of onion root tip and place in a microcentrifuge tube labeled “C” for the control condition.
   • Add 2 µL of the 5 mg/mL nocodazole to 1 mL of water in the microcentrifuge tube labeled N and 1 mL of water only to the tube labeled C.
   • Leave the tubes containing the root tips to incubate for 12 hours and wipe down the workspace with 70% ethanol.
   • When incubation is complete, set a heat block to 60 ºC.
   • Wash the nocodazole treated tips with water and then aspirate the liquid.
   • Repeat this wash two more times, for a total of three washes.
   • Fill both tubes with 1 mL of one normal hydrochloric acid. Incubate the tubes in a 60 ºC heat block for 12 - 15 min, then discard the acid into a waste flask.
   • Now, add 1 mL of distilled water drop wise to the tubes at least three times to rinse the samples.
   • Now add one microliter of DAPI stain to 10 mL of phosphate buffered saline (PBS).
   • Then place 50 µL of this dilute DAPI stain into each tube, and leave them at room temperature to incubate for 12 min.
   • After this, preform three distilled water washes as in step 10.
   • Label one microscope slide as 'Control' and another as 'Nocodazole'.
   • Transfer the stained roots onto their respective microscope slides and add a drop of water to the control slide and a drop of nocodazole to the nocodazole slide.
   • Use the razor blade to remove the unstained portions of each root tip and place a glass cover slip over each sample.
   • Carefully press down on each cover slip with a gloved fingertip, and then let the slides sit for 10 min.
   • Finally, view the slides under the 40X objective of a fluorescence microscope and capture images of your root tip cell preparations.
2. Understanding the Loss of Cell Cycle Control Expand
   • NOTE: Before cells can pass through all of the stages of the cell cycle there are several checkpoints they must go through. These checkpoints are regulated by cyclin and cyclin-dependent kinase proteins, or CDKs. If the cyclins and CDKs determine that previous cellular events have not been adequately completed they will arrest the cells at this step and prevent them from proliferating. In cancer cells one or several of these molecular restraints for regulating cell division are lost and this allows the damaged cells to escape from cell proliferation control. These abnormal cells develop into tumors which are masses of uncontrolled proliferating cells that interfere with the normal functions of the body. Notice how before it divides this cancer cell rounds up and refracts light very strongly under the light microscope. This is because not all cancer cells successfully divide into daughter cells after each round of mitosis. This will mean that they often contain an excess of organelles and DNA and this increase in cellular content means they will refract light more intensely than normal cells do.
3. Results Expand
   • Examine your collected images and record in Table 1 the different cell structures and stages that could be identified in the onion tip slice that was treated with nocodazole. Click Here to download Table 1
   • If there are any cells undergoing mitosis, note what stages of cell division you see.
   • Record the percentage of cells in each stage of mitosis in Table 1.
   • Next, examine the images of the untreated onion tip slice, and record what cell structures can you identify.
   • If there are any cells undergoing mitosis, consider what stages of cell division you see, and record the percentages of cells in each stage of mitosis in the Table 1.

The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a chemical that interferes with microtubular polymerization, all of the cells will be arrested at the same stage of the cell cycle and that in untreated onion tip slices all of the different stages of the cell cycle will be visualized. The null hypothesis is that there will be no differences in mitosis between the two groups and that different stages of the cell cycle will be visualized. To prepare for this experiment you will use an onion that has been placed in water for several days until new white root tips have begun to sprout.

Use a razor blade to cut a one centimeter piece from the end of one of the root tips. Place the slice of onion into a microcentrifuge tube labeled N for the nocodazole condition. Then cut a second one centimeter piece of onion root tip and place in a microcentrifuge tube labeled C for the control condition.

Add two microliters of five milligram per milliliter nocodazole to one milliliter of water in the microcentrifuge tube labeled N and one milliliter of water to the tube labeled C.Leave the tubes containing the root tips to incubate for 12 hours and wipe down the workspace with 70%ethanol. When incubation is complete set a heat block to 60 degrees Celsius. Wash the nocodazole treated tips with water and then aspirate the liquid.

Repeat this wash two times for a total of three washes. Fill both tubes with one milliliter of one normal hydrochloric acid. Incubate the tubes in a 60 degree Celsius heat block for 12 to 15 minutes then discard the acid into a waste flask and add one milliliter of distilled water drop wise to the tubes at least three times to rinse the samples.

Now add one microliter of DAPI stain to 10 milliliters of phosphate buffered saline. Then place 50 microliters of the dilute DAPI stain into each tube and leave them at room temperature to incubate for 12 minutes. After this preform three distilled water washes as just demonstrated.

Label one microscope slide as Control and another as Nocodazole. Transfer the stained roots on to their respective microscope slides and add a drop of water to the control slide and a drop of nocodazole to the nocodazole slide. Use the razor blade to remove the unstained portions of each root tip and place a glass cover slip over each sample.

Carefully press down on each cover slip with a gloved finger tip and then let the slides sit for 10 minutes. Finally view the slides under the 40x objective of a light or fluorescent microscope and capture images of your root tip cell preparations. Before cells can pass through all of the stages of the cell cycle there are several checkpoints they must go through.

These checkpoints are regulated by cyclin and cyclin-dependent kinase proteins, or CDKs. If the cyclins and CDKs determine that previous cellular events have not been adequately completed they will arrest the cells at this step and prevent them from proliferating. In cancer cells one or several of these molecular restraints for regulating cell division are lost and this allows the damaged cells to escape from cell proliferation control.

These abnormal cells develop into tumors which are masses of uncontrolled proliferating cells that interfere with the normal functions of the body. Notice how before it divides this cancer cell rounds up and refracts light very strongly under the light microscope. This is because not all cancer cells successfully divide into daughter cells after each round of mitosis.

This will mean that they often contain an excess of organelles and DNA and this increase in cellular content means they will refract light more intensely than normal cells do. Now that all of the data has been collected let's look at our results. What cell structures can be identified in the onion tip slice that was treated with nocodazole?

Are any cells undergoing mitosis? What stages of cell division do you see? Record the percentage of cells in each stage of mitosis in the table.

Next look at the untreated onion tip slice. What cell structures can you identify? Are any cells undergoing mitosis?

What stages of cell division do you see? Record the percentages of cells in each stage of mitosis in the table. You will have noticed that most of the cells in the nocodazole treated onion slice are in the same stage of mitosis due to the microtubule disrupting properties of the reagent which prevent the formation of the spindle fibers and subsequent chromosome segregation and cell division.

Many different stages of mitosis are visible in the cells of the untreated slice however as mitosis is an ongoing process in healthy organisms.