ASSET Student Laboratory

Transcription

1 ASSET Student Laboratory The Effect of Cigarette Smoke and Alcohol on Tetrahymena Background Information While the general effects of smoking and alcohol use on humans are well documented, it is useful to further investigate what s happening at the cellular level. We know that it is not healthy to smoke in any amount or to over-indulge in alcohol, but what specific changes can be caused by these activities? It s obviously not possible or advisable to run experiments like this on humans, so a suitable proxy must be found. In many laboratories, other mammals such as monkeys or rats are chosen because they have similar genetics and body structures. Trials involving mammals are, however, expensive and very tightly regulated. Fortunately, in some cases the single cell protozoan Tetrahymena can be used as an experimental stand in for humans. At first glance, humans and Tetrahymena don t seem very similar at all. They belong to different kingdoms. Humans are made up of trillions of cells (and hundreds of different types!), while Tetrahymena is composed of only one cell. Nonetheless, some aspects of Tetrahymena biology are remarkably similar to basic human biology. One biological similarity that we are going to use in this lab is that Tetrahymena and humans both have cilia. Cilia are small, hair-like structures that are found on the outside of Tetrahymena and on many different types of cells in humans. Many cilia are flexible and move back and forth. These are called motile cilia. Each single cilium consists of pairs of microtubules arranged in a 9+2 configuration. That is, there is a ring of nine pairs of microtubules around the outside of the cilia, and one pair in the middle. A cross section of several stained cilia displaying the classic 9+2 arrangement is seen in Figure 1. Microtubules, such as those that form the 9+2 arrangement of cilia, are made up of a protein pair of microtubules Figure 1. Cilia cross-section showing 9+2 arrangement of microtubules. called tubulin. A protein called dynein provides the force needed to move the microtubules, and that action in turn moves the cilium. Dynein is attached to one microtubule in each pair of microtubules that makes up the outer ring (the 9 in the 9+2 arrangement) of the cilia. Dynein is known as a molecular motor, because it can change its shape when it binds to ATP or when the ATP is hydrolyzed and released. The shape change that the protein undergoes allows it to bind to the second microtubule in the pair, pull on it, and then release. It s very much like a group of rowers all pulling on the same oar; they pull against the water to move the boat, remove the oar from the water and reset it for another pull, then dip in again. In this way, dynein can pull and release, pull and release, causing the cilia to bend. This requires thousands of molecules of ATP every second, and a way to coordinate each pair of microtubules, so that they all beat in the same direction at the same time. The mechanism of ciliary coordination is not yet fully understood. The best-known ciliated cells in the human body are in the respiratory system. In the bronchi, ciliated cells, all moving their cilia in the same direction, move mucus and debris up and out of the respiratory 1

2 system, sort of like garbage removers. The reproductive systems of both males and females also contain cilia; the oviduct in females and several ducts in the testes of males have these structures. In females, cilia move eggs into the Fallopian tubes where they can be successfully fertilized, and then into the uterus where it s safe for the egg to implant and grow into a new human being. In males, cilia move developing sperm cells along until they develop tails and are able to move on their own. Ciliated cells are also found parts of the brain and spinal cord to help circulate fluid around the central nervous system. In Tetrahymena, motile cilia propel the cell through its water environment, and also help sweep food into the cell s mouth (oral apparatus). Another class of cilia exists. They are called primary cilia and are not motile. They have a structure similar to motile cilia, but lack the center pair of microtubules, and so are said to have a 9+0 arrangement. These cilia do not move, but are present on almost all types of cells in the human body. They are thought to be used for communication between cells, and may even play roles in several serious diseases. Strains of rats that do not have primary cilia have been developed. Without these cilia, their embryos fail to develop properly. The fact that embryos fail to develop in the absence of these primary cilia indicates that they probably play an important role in cell-to-cell communication. In adult rats, a disorder known as polycystic kidney disease develops, indicating that cells with primary cilia are probably involved in the disease somehow. Although humans and Tetrahymena belong to two separate kingdoms, the structure of their cilia and the proteins from which they are made are the same. This makes Tetrahymena an excellent stand-in for human ciliated cells in research. Since cilia play an important role in many different cell types and body systems, scientists are interested in studying how they are affected by common toxins. Cigarette smoke and alcohol are among the most common toxins to which humans voluntarily expose themselves. Studying the effects of alcohol is pretty straightforward because alcohol is a single chemical substance. (Different forms of alcoholic beverages, of course, have different extra substances, but it s generally only the alcohol in the drink that causes the damaging effects.) Alcohol affects biochemical pathways that are essential to proper ciliary function. At first exposure, cilia are stimulated to beat faster by low levels of alcohol, but with higher levels over a longer time, they slow down and fail to function. Smoking is more difficult to examine, because the smoke from cigarettes contains thousands of different chemicals. These range from tar, nicotine and acetone to nickel and formaldehyde. Because there are so many chemicals, it would be very difficult to pinpoint one specific location in a cell that is affected; many different cellular pathways will be changed in some way. In this lab you will examine the effects of cigarette smoke extract and alcohol on living Tetrahymena, observing effects on swimming behavior, speed, shape, or other movement. Based on your observations, you will consider whether it is likely that cigarette smoke extract and alcohol may have an effect on ciliary activity and possibly human health. Materials per group 1 petri dish of Tetrahymena culture 1 P200 micropipette 1 vial 10 mm Tris solution 1 box micropipette tips 1 vial cigarette smoke extract microscope slides 1 vial alcohol sample 1 marker 1 vial non-alcohol sample 1 timer 4 microfuge tubes 1 microfuge tube rack 2

3 Procedure Part 1: Expose cells to the substances. A. Alcohol 1. Take 2 microfuge tubes and label one Beer and the other Control. Put your group initials on the tube. 2. Set the P200 to 150!L. Using a new tip, add 300!L of cells to each tube. (Pipette twice to get a total of 300!L of cells.) You don t have to change the tip for each tube because you re pipetting the same culture into empty tubes, so the tip won t become contaminated. 3. Eject your tip into the waste beaker. 4. Using a new pipette tip, add 300!L of beer to the Beer tube. Close the cap. 5. Eject your tip into the waste beaker. 6. Using a new pipette tip, add 300!L of non-alcoholic beer to the Control tube. Close the cap. 7. Eject your tip into the waste beaker. 8. Start your timer and let the cells incubate at room temperature for 5 minutes. 9. While you wait for your cells to incubate, take a microscope slide and using a marker, draw a line down the middle of the slide as illustrated below. 10. In the corners of the slide, label C for control and B for beer as illustrated below. C B 11. Re-set your micropipette to 20!L and put on a fresh tip. After 5 minutes, place 20!L of your control on the C side of your microscope slide. Change tips and place 20!L of your beer mixture on the B side. 12. You do not need to use a cover slip. This arrangement will make it easy to cruise back and forth between the droplets and compare them. 13. Observe the drops and record your observations in Table 1. Watch for changes in swimming behavior, speed, shape, or other movement. 3

4 B. Smoke Extract 1. Take 2 microfuge tubes and label one Smoke and the other Control. Make sure you put your group initials on the tube as well. 2. Set the P200 to 150!L. Using a new tip, add 300!L of cells to each tube. (Pipette twice to get a total of 300!L of cells.) You don t have to change the tip for each tube because you re pipetting the same culture into empty tubes, so the tip won t become contaminated. 3. Eject your tip into the waste beaker. 4. Using a new pipette tip, add 300!L of smoke extract to the Smoke tube. Close the cap. 5. Eject your tip into the waste beaker. 6. Using a new pipette tip, add 300!L of 10 mm Tris solution to the Control tube. Close the cap. (Tris is a mixture of water and some other substances that make the solution isotonic to the cells so they won t swell or shrivel up. It s almost the same as using water for a control.) 7. Eject your tip into the waste beaker. 8. Start your timer and let the cells incubate at room temperature for 5 minutes. 9. While you wait for your cells to incubate, take a microscope slide and using a marker, draw a line down the middle of the slide as illustrated below. 10. In the corners of the slide, label C for control and S for smoke extract as illustrated below. C S 11. Re-set your micropipette to 20!L and put on a fresh tip. After 5 minutes, place 20!L of your control on the C side of your microscope slide. Change tips and place 20!L of your beer mixture on the B side. 12. You do not need to use a cover slip. This arrangement will make it easy to cruise back and forth between the droplets and compare them. 13. Observe the droplets and record your observations in Table 2. Watch for changes in swimming behavior, speed, shape, or other movement. 4

5 Cleaning Up The Effect of Cigarette Smoke and Alcohol on Tetrahymena 1. After you are finished making your observations for both the beer and smoke extract samples, rinse the microfuge tubes labeled B, C, S and C and the microscope slides you used in your experiment. Tap the tubes on a paper towel to get as much water out as possible and leave them in the rack for the next class. Follow your teacher s instructions with the slides. 2. Wash your hands with soap and water. Data Tables Table 1. Effects of beer on Tetrahymena. Treatment Non-Alcoholic Beer (Control) Beer Observations Table 2. Effects of smoke extracts on Tetrahymena. Treatment Type Control (no Smoke Extract) Smoke Extract Observations Computer Tracking Option If your teacher directs you to, before you dispose of your slide, gather data to look at tracking. 1. Using the software on your computer, take a 10-second video of each droplet. You should try to find a spot on your slide where there aren t too many cells on your screen. You want to get a good sample size without having the cells swim over each other too often. 5

6 2. When you have taken the videos, save them in a location from which you can easily access them (e.g. thumb drive, network drive, etc.). Make sure you give each file an appropriate name. Don t name it Jimmy s Tetrahymena, because that doesn t describe what is going on, and your name may not be Jimmy. JS smoke extract or JS smoke control is much more descriptive (where JS are your initials). 3. Using the video at the ASSET website as a guide, analyze your video using ImageJ Compare the tracks that you see created on the screen. Record the approximate percent direction change on your data table. This unit is defined as the percentage of cells that deviated from a straight path (by 17 or more) at least once in one second of swimming. Treatment Type Control (No Smoke Extract) Smoke Extract Control (Nonalcoholic Beer) Alcoholic Beer % Direction Change Conclusion Questions 1. What body systems or organs do you think might be affected by alcohol or smoking? 2. What are cilia and what are they made of? 3. What do single-celled organisms like Tetrahymena use their cilia for? 4. Many cells in multicellular organisms have cilia. What are some things they could be used for? 5. What is a primary cilium and how is it different from the cilia mentioned above? 6. What chemicals in cigarette smoke might have an effect on how cilia move? 6

7 7. If the cilia in your respiratory tract were damaged by substances in cigarette smoke, making them immotile (unable to move), how might that make it more likely that you would develop lung cancer? Answer this by responding to the questions below: a. What is the function of normal cilia in a healthy lung? b. Why is it important for cilia to do this? c. Why might it be a problem for the lungs if the cilia stop doing their job? 8. What chemical(s) in beer might have an effect on how cilia move? 9. How can you tell that the movement of the Tetrahymena cilia has been changed without being able to see individual cilia? 10. What allows you to conclude that the effects that you observed with beer were actually caused by the alcohol and not something else, like carbohydrates or protein? 11. Do you think the results of a study involving Tetrahymena cilia could provide information helpful to understanding cilia-related human diseases? Why or why not? 12. Most alcoholics (80-95% of them) also smoke. Why does this put them at particular risk for both lung cancer and infections like pneumonia that are caused by bacteria? 7