What is nocodazole and how does it affect cells in a cell cycle experiment?

Nocodazole is a chemical that interferes with microtubular polymerization, preventing microtubules from forming properly. When applied to root tip cells, nocodazole arrests all cells at the same stage of the cell cycle by disrupting the spindle apparatus needed for mitosis. This allows researchers to observe cells frozen at a particular division stage rather than seeing a mixed population.

Why do cancer cells refract light more intensely than normal cells under a microscope?

Cancer cells often contain an excess of organelles and DNA because not all cancer cells successfully divide into daughter cells after mitosis. This increased cellular content causes them to refract light more intensely than normal cells. The cells also round up during division, further increasing light refraction compared to typical cell morphology.

What role do cyclins and CDKs play in controlling cell division?

Cyclins and cyclin-dependent kinase proteins, or CDKs, regulate cell cycle checkpoints by monitoring whether previous cellular events have been completed adequately. If they detect incomplete processes, they arrest cells at that checkpoint and prevent proliferation. In cancer cells, these molecular restraints are lost, allowing damaged cells to escape control and develop into tumors.

How does DAPI staining help visualize cell cycle stages in root tip preparations?

DAPI is a fluorescent dye that binds to DNA, making cell nuclei and chromosomes visible under a fluorescence microscope. By staining root tip cells with diluted DAPI and viewing them at 40X magnification, researchers can identify different mitotic stages based on chromosome arrangement and nuclear morphology. This allows accurate counting and classification of cells in each stage of division.

What is the difference between the control and nocodazole-treated samples in this experiment?

The control sample contains untreated onion root tip cells, showing all different stages of the cell cycle distributed naturally. The nocodazole-treated sample has cells arrested at the same division stage due to microtubule disruption. Comparing these samples demonstrates how chemical interference affects cell cycle progression and reveals the importance of microtubules in mitosis.

Why are onion root tips ideal tissue for observing the cell cycle?

Onion root tips contain rapidly dividing cells with high mitotic activity, making it easy to observe multiple cell cycle stages in a single preparation. The cells are small and transparent, allowing clear visualization under a microscope. Root tips also grow continuously when placed in water, providing fresh tissue with abundant cells undergoing active division.

How does hydrochloric acid treatment prepare root tip cells for microscopy?

Hydrochloric acid treatment softens and partially digests the cell wall and middle lamella of root tip cells, making them easier to separate and spread on a microscope slide. Heating the acid to 60 degrees Celsius accelerates this process. This preparation step allows individual cells to be visualized clearly without cell wall interference, enabling accurate observation of chromosomes and mitotic stages.

Cell Division

Concepts Instructor Prep Student Protocol

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The cell cycle refers to the sequence of events throughout a typical cell's life - involving growth, DNA replication, and preparation for cell division. For sexually-reproducing organisms, life begins as a zygote, a fertilized egg. Over time, that original single cell grows and divides in a controlled manner to produce a multicellular, complex individual. Cells continue this process, especially to maintain and repair tissues during aging.

Now let's take a closer look. In eukaryotes, double-stranded DNA is specially organized within a membrane-bound nucleus to accommodate the cell's limited space. At the first level of compaction, DNA is wrapped tightly around specific proteins called histones. This combination of DNA protein particles is then repeated and packed into arrays known as nucleosomes, which, along with linker DNA, form coiled chromatin fibers. Finally, additional fibrous proteins compact the chromatin even further, packing long lengths of DNA into tightly-condensed units. Recognized as chromosomes, depending on the phase of cell division.

To prepare for division, cells must go through interphase, which is divided into three stages. G1, S, and G2. In G1, the first gap phase, a newly-generated daughter cell grows in size and prepares for DNA duplication in the next phase. Now in S, the synthesis phase, cells duplicate their nuclear DNA, which remains packaged as chromatin. Cells also duplicate the centrosomes, the microtubule-organizing structures which form the mitotic spindle apparatus. Finally, in G2, the second gap phase, cells continue to grow, multiply organelles and proteins that are required for mitosis, and replenish their energy stores. The cell is now ready to enter the first stage of mitosis.

Comprised of five unique stages, mitosis is a form of division where a cell's genetic material is partitioned between two daughter cells. First during prophase in humans, nucleic chromatin condenses into X-shaped chromosomes, composed of sister chromatid pairs attached at centromere junctions. Concurrently outside the nucleus, centrosomes migrate to opposite sides of the cell. As they do so, microtubule rods begin to grow from each. Either towards the cell's interior or exterior, forming a web-like spindle apparatus. Next, the nuclear envelope dissolves during prometaphase, exposing the chromosomes to the cell's other contents. Protein structures also appear on both sides of the centromeres, one for every chromatid. Once these kinetochores form, extending interior microtubules fasten to them, with each sister chromatid being tethered to a different pole.

Mitosis then progresses to metaphase, where the spindle apparatus rearranges the chromosomes so that they are similarly oriented in a fixed row along the cell's equator. During anaphase, kinetochore-affixed microtubules shorten. And sister chromatids, now individually referred to as chromosomes, are dragged apart. These and other microtubule dynamics also elongate the cell. Finally, the chromosomes land at opposite cell sides during telophase. And the spindle apparatus disbands. The genetic material loosens, and two nuclear envelopes, one around each chromosome set, arise. During telophase, a distinct process, not technically a stage of mitosis, called cytokinesis, also divides the cell. Thus, the end result of mitosis is a cell pair genetically identical to their precursor.

Unfortunately, mutations can cause damage to genes controlling cell cycle regulation, which leads to cell division proceeding unchecked. In this case, each successive cell division produces daughter cells with even more damage…and the faulty growth regulation ultimately leads to the formation of cell masses called tumors.

In this lab, you will examine the different stages of mitosis using onion root tip cells. And then examine what happens when cell cycle control is lost.

Print Procedure Steps

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Observing the Cell Cycle in a Root Tip
- 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.
Understanding the Loss of Cell Cycle Control
- 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.
Results
- 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.

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