Lab 4: Cell Division – Mitosis and Meiosis
How do eukaryotic cells divide to produce genetically identical cells or to produce gametes with half the normal DNA?
Background:
One of the characteristics of living things is the ability to replicate and pass on genetic information to the next generation. Cell division in individual bacteria and archaea usually occurs by binary fission. Mitochondria and chloroplasts also replicate by binary fission, which is evidence of the evolutionary relationship between these organelles and prokaryotes.
Cell division in eukaryotes is more complex. It requires the cell to manage a complicated process of duplicating the nucleus, other organelles, and multiple chromosomes. This process, called the cell cycle, is divided into three parts: interphase, mitosis, and cytokinesis. In the first growth phase (G1), the cell grows and prepares to duplicate its DNA. In the synthesis phase (S), the chromosomes are replicated. In the second growth phase (G2), the cell prepares to divide. In mitosis, the duplicated chromosomes are separated into two nuclei. In most cases, mitosis is followed by cytokinesis, when the cytoplasm divides and organelles separate into daughter cells. This type of cell division is asexual and is important for growth, renewal, and repair of multicellular organisms.
Cell division is tightly controlled by complexes made of several specific proteins. These complexes contain enzymes called cyclin-dependent kinases (CDKs), which turn on or off the various processes that take place in cell division. CDK partners with a family of proteins called cyclins. One such complex is mitosis-promoting factor (MPF), sometimes called maturation-promoting factor, which contains cyclin A or B and cyclin-dependent kinase (CDK). CDK is activated when it is bound to cyclin, interacting with various other proteins that, in this case, allow the cell to proceed from G2 into mitosis. The levels of cyclin change during the cell cycle. In most cases, cytokinesis follows mitosis.
As shown in Figure 2, different CKs are produced during the phases. The cyclins determine which processes in cell division are turned on or off and in what order by CDK. As each cyclin is turned on or off, CDK causes the cell to progress through the stages in the cell cycle.
Cyclins and CDKs do not allow the cell to progress through its cycle automatically. There are three checkpoints a cell must pass through the G1 checkpoint, G2 checkpoint, and the M-spindle checkpoint. At each of the checkpoints, the cell checks that it has completed all of the tasks needed and is ready to proceed to the next step in its cycle. Cells pass the G1 checkpoint when they are stimulated by appropriate external growth factors for example, platelet-derived growth factor (PDGF) stimulates cells near a wound to divide so that they can repair the injury. The G2 checkpoint checks for damage after DNA is replicated. And if there is damage, it prevents the cell from going into mitosis. The M-spindle (metaphase) checkpoint assures that the mitotic spindles or microtubules are properly attached to the kinetochores (anchor sites on the chromosomes). If the spindles are not anchored properly, the cell does not continue on through mitosis. The cell cycle is regulated very precisely. Mutations in cell cycle genes that interfere with proper cell cycle control are found very often in cancer cells.
Figure 3 illustrates how the chromosomes move during mitosis. It is important for you to model how the duplicated chromosomes align, separate, and move into new cells.
The stages of mitosis:
v Prophase. During prophase, the chromosomes supercoil and the fibers of the spindle apparatus begin to form between centrosomes located at the pole of the cells. The nuclear membrane also disintegrates at this time, freeing the chromosomes into the surrounding cytoplasm.
v Prometaphase. During prometaphase, some of the fibers attach to the centromere of each pair of sister chromatids and they begin to move toward the center of the cell.
v Metaphase. At metaphase the chromosomes have come to rest along the center plane of the cell.
v Anaphase. During anaphase, the centromeres split and the sister chromatids begin to migrate toward the opposite poles of the cell.
v Telophase. During telophase, the chromosomes at either end of the cell cluster begin to cluster together, which facilitates the formation of a new nuclear membrane. This also is when cytokinesis occurs, leading to two separate cells. One way to identify that telophase has begun is by looking for the formation of the cell plate, the new cell wall forming between the two cells.
Learning Objectives: The objectives of this lab exercise are for you to
• Better understand the process and stages of mitosis.
• Apply an analytical technique by which the relative length of each stage of mitosis can be estimated.
What you will do
Today, you will use the compound microscope to observe onion root tip cells and test hypotheses about mitosis. You will apply simple statistical tests to make inferences based on your hypotheses. Additionally, you will continue to refine your skills in using microscopes, making slide preparations, and data analysis.
Laboratory Objectives
As a result of participating in this laboratory activity, you will:
• Develop a hypothesis
• Analyze data using statistics
• Draw conclusions based on gathered data
• Use appropriate terminology when discussing mitosis and integrate your knowledge of mitosis into the cell cycle
• Model the process of mitosis
Cell Cycle – How long does a cell spend in each phase of mitosis?
Determine how long a cell spends in each phase of mitosis (Interphase (which includes G1, S, G2), Prophase, Metaphase, Anaphase, and Telophase). Complete this project with your lab group, and clearly write up your investigation in your lab guidebook. Parts 1‐3 should be included in the data and field notes section. Part 4 should be in your data analysis and results section. Part 5 should be included in the conclusions section.
1. Develop a hypothesis for the percentage of time a cell spends in each phase of the cell cycle.
2. Make a prediction about your results that is based on your hypothesis. For example, if you were given 100 onion cells, and your alternative hypothesis is assumed to be true, how many of those 100 cells would be found in each of the stages of the cell cycle (that is, interphase, prophase, metaphase, anaphase and telophase)? These are your expected values based on your hypothesis.
3. Develop a method to test your hypothesis using professionally stained slides (these are easier for observing chromosomes). Record your method such that others could replicate your experiment.
4. Collect your data. Construct a table to organize your raw data. Be sure to include appropriate titles and labels. Use the professionally‐prepared slides of onion root tips to sample the cells using the method you developed.
5. Analyze your results. Conduct a Chi‐squared statistical test on your collected data (see Appendix C). Organize your results as a pie‐chart. Don’t forget to include a title and other appropriate labels.
6. Draw conclusions. Discuss and interpret what your results mean, including your statistical tests. Be sure to go back to your hypothesis to make a clear link between your hypothesis and your results.
Post Lab. Arrange your data, analyses and conclusions using the following headers and respective items under each heading.
1. Raw Data, Field Notes and Observations (5 pts)
*** Clearly labeled sketches of 5 cells (from the professionally stained slide) with the location and arrangement of the chromosomes clearly marked, and the stages of mitosis identified.
*** Record your hypothesis, prediction and method (#1‐3).
*** Construct a table to organize your raw data. Include titles and labels.
2. Data Analysis and Results (5 pts)
*** Include all your work (i.e., calculations) for calculating Chi‐square.
*** Organize your data into a pie‐chart. Include labels and figure legends.
3. Conclusions (5 pts)
*** Discuss and interpret what your results mean, including results of your statistical test.
4. Reflection (5 pts)
*** What is the purpose of mitosis?
*** What might happen if your cells did not undergo mitosis?
Appendix A. Cells in Mitosis Photographs
In prophase, the chromatin has begun to gather together and form into chromosomes. Notice especially nuclear size and density. A cell in interphase has a nuclear membrane, but the one in the prophase cell has broken down.
Prophase
At metaphase, the chromosomes have begun to congregate in the center of the cell, between the poles, in an area called the equator. The identical chromosomes are being prepared for migration to the opposite sides of the cell toward the poles.
Metaphase
Anaphase is characterized by the migration of chromosomes toward the poles. Each set of chromosomes that you see in the center of the slide is an exact duplicate of the other. The two cells that will eventually form from this separation are thus duplicates, each with the same amount and quality of DNA.
Anaphase
During telophase, cytokinesis begins and a new cell wall forms at what was once the equator. In plant cells the cell wall forms in the center of the cell and is built outward until it reaches the periphery. Once the periphery is reached, the cell has completed the period and G1 begins.
Telophase
Appendix B. Chi‐Square Statistic
Today, we will be using statistics to test hypotheses, through the Chi‐Square (χ2) Test Statistic. This test compares actual (observed) and predicted (expected) outcomes of an experiment. For each type of outcome (in our case, the phases in mitosis) in the experiment, there are both observed and expected numbers of occurrences. The Chi‐square statistic is used in this test:
.
Statisticians have determined the probability distribution of the Chi‐square statistic. You will use Table 1 for the Chi‐square statistic to analyze your results. This table will let you decide if the
difference between observed and expected values is small enough to be due to chance (meaning the hypothesis may be true) or is too large to be due to chance (meaning the hypothesis produces a poor prediction of your results and is unlikely to be true).
Table 1. Chi Square Probability TableDegrees of freedom / Reject the hypothesis if Χ2 is greater than:
1 / 3.841
2 / 5.991
3 / 7.815
4 / 9.488
5 / 11.070
Table 2. A simple way to manage the Chi‐Squared statistic calculations
Stage of Observed (O) # Expected (E) # (O‐E) (O‐E)2 (O‐E)2/E
Mitosis
Interphase
Prophase
Metaphase
Anaphase
Telophase
‐‐‐‐‐ / ‐‐‐‐ / ‐‐‐ / ‐‐‐ / SUM this column to get Chi‐SquareIn Table 2, the observed numbers will come from the data you collect during lab while the expected numbers will be derived from your hypotheses. To determine the degrees of freedom (df) you simply take the number of phases in mitosis (or rows in the table) and subtract 1. In our case, df = 5‐1 = 4.
Now use Table 1 to look at the row for the appropriate degrees of freedom (in our case, 4) to get a Chi‐ Squared probability value. Compare this number to the value you obtained in Table 2 and determine whether or not the hypothesis you were testing holds true. If the number you calculated is greater than the number you pulled from Table 1, you fail to accept (i.e. reject) the hypothesis you were testing. However, if the value you calculated for Chi‐Squared is smaller than the number in Table 1, you will not reject the hypothesis you tested.