BISC110/F12: Series 1 Lab 4 Student-designed Exp1

Series 1 Lab 4 Variable Testing in Tetrahymena

Adapted from Bozzone, M.D., and D.A. Martin 2000. An experimenal system to study phagocytosis. Pages 405-415, in Tested studies for laboratory teaching, Volume 21 (S.J. Karcher, Editor). Proceedings of the 21st Workshop/Conference of the Association for Biology Laboratory Education (ABLE).

You will have the opportunity to carry out an experiment that you and your partner design. Each group of four students will focus on one variable.

I. Investigating Phagocytosis in Tetrahymena pyriformis

You will have the opportunity to test a particular aspect of phagocytosis in Tetrahymena pyriformis that interests you. Gronlien et al. (2002) propose that the microstomal form of a particular species of Tetrahymena (not the species we are studying) exhibit non-specific phagocytosis. You might choose to see if that is also true of Tetrahymena pyriformis. There is also previous research that concludes that there is a particle size preference for phagocytosis in Tetrahymena. You might choose to explore that idea.

Many of the research papers that explore phagocytosis in Tetrahymena propose that the cytoskeleton, particularly the actin containing elements of the cytoskeleton, are crucial to some or all of the parts of phagocytosis in Tetrahymena. There seems to be some conflicting evidence addressing the role of the tubulin containing part of the cytoskeleton. You could choose to explore either the role of microfilaments (actin) or microtubules (tubulin) in phagocytosis in Tetrahymena pyriformis.

Main Topic Choices:
Is phagocytosis in Tetrahymena pyriformis selective or non-selective for particle size or type? (Study phagocytosis of Polybead dyed microspheres that vary in color and size OR study phagocytosis of microspheres and India ink)

What role does the cytoskeletal element actin (in microfilaments & cilia) play in one or more aspects of phagocytosis (either engulfment, phagosome formation &/or filling, movement or vesicle fusion) in Tetrahymena pyriformis? (Study phagocytosis of India ink in the absence and presence of the actin inhibitor cytochalasin B)

What role does the cytoskeleton element tubulin (in microtubules) play in some aspect of phagocytosis (either engulfment, phagosome formation, filling, movement or fusion) in Tetrahymena pyriformis? (Study phagocytosis of India ink in the absence and presence of the tubulin inhibitors colchicine or nocodazole)

Stock concentrations of reagents available to you:

In addition to the reagents that you used previously, cytoskeleton inhibitors and Polybead dyed microspheres (different colors and sizes) will be available for this experiment. CAUTION: Wear gloves and exercise caution when handling the inhibitors because they are toxic.

Cytochalasin B 14mM (6.7mg/ml) in DMSO
Colchicine 25mM (10mg/ml) in water
Nocodazole 2mg/ml (7mM) in DMSO
India ink (Speedball, Product #3328, #3338, Statesville, NC), according to the manufacturer, contains (vol./vol.): water (75-85%), carbon black (7-9%), shellac (6-8%), ammonium hydroxide (1-2%), phenol (0.45%) and ethylene glycol (1.6%). The carbon black is dispersed as ~ 20nm carbon particles but it ranges, in the commercial grade used for India ink from 13-75nm (0.013-.075µm) or larger when aggregated.

Polybead Dyed Microspheres available:
0.2µm Blue
0.5µm Blue
1.0µm Blue
3.0µm Blue
6.0µm Blue
10.0µm Blue
0.5µm Yellow
3.0µm Yellow
6.0µm Yellow
Polybeads are packaged as 2.5% aqueous suspension (water) and will be diluted to a working concentration of 0.08% prior to lab. This concentration approximately equals the working concentration of carbon particles in 1% India ink.

ONE POSSIBLE PROTOCOL TO TEST THE ROLE OF ACTIN OR TUBULIN IN PHAGOCYTOSIS (can be modified)

1. In a microfuge tube, make a 1:100 dilution of the drug inhibitor in Tetrahymena stock by combining 99 µl Tetrahymena (grown for 48hrs in 2% proteose-peptone) and 1 µl inhibitor. (Do not add more volume of inhibitor because some of the inhibitors are diluted in toxic compounds that could negatively affect the viability of your cells at higher concentrations.) Include an appropriate control for your experiment to make sure that your inhibitor's diluent is not affecting vacuole formation. If your inhibitor is disolved in DMSO, your control should be 1 µl of DMSO added to 99 µl of Tetrahymena.
2. Incubate for 0-10 minutes if you think your inhibitor needs time to affect the element (actin, tubulin, or whatever you are trying to assess the role of in phagocytosis). Note that you can omit this pre-incubation step or shorten it if your research indicates that your inhibitor acts immediately on the element you are testing. What is the effective concentration of inhibitor in this important step?
3. Add 100 µl of 1% India ink to the 100 µl Tetrahymena mixture prepared in step 1 in a microfuge tube. You will probably want to remove a 20 microliter sample immediately in order to assess a "zero time" control (see step 4). Calculate the concentration of your inhibitor and India ink after this combination. Note that you are making another ~1:100 dilution of your inhibitor in this step.
4. At several time points (0, 15, 30 min. are suggested but you may vary this), REMOVE a 20 µl aliquot from the reaction microfuge tube of Tetrahymena/inhibitor/ink and the control tube of Tetrahymena/diluent/ink and, IN THE HOOD, put each aliquot in a clean, labeled microfuge tube containing 10 µl of 3% gluteraldehyde. This will stop phagocytosis by fixing the cells so you can study them later. Incubate for one minute then add 2 ul of 2.5M glycine. Incubate for one minute. If you would prefer to study your cells live, you will have to take your measurements very quickly to keep the timing relatively accurate. What is the effective concentration of the gluteraldehyde fixative?
5. Make a slide for each time point IN THE HOOD by placing 20 µl of each tube from step 4 on a separate slide and cover each with a cover slip. Don’t make all your slides at the same time or the Tetrahymena will dry out before you can count the filled vacuoles.
6. Count or assess whatever you have decided to measure in as many cells as you can for each time point.
   
   

ONE POSSIBLE PROTOCOL TO TEST THE ROLE OF PARTICLE SIZE IN PHAGOCYTOSIS (can be modified)
There are many different approaches you might take to determine the affect of particle size on phagocytosis. You might want to determine the ability of Tetrahymena to phagocytose different particle sizes individually. For example, you might test the ability of Tetrahymena to phagocytose the 0.2µm blue, 0.5µm blue, 1.0µm blue, 3.0µm blue, 6.0µm blue, and 10.0µm blue Polybead dyed microspheres by exposing the Tetrahymena to just one of these blue microspheres at a time. Alternatively, you might be interested to know whether Tetrahymena are selective for particle size when exposed to microspheres of two different sizes at once. To address this question, you might expose Tetrahymena to the 3.0µm blue and 6.0µm yellow microspheres at the same time. Compared to counting ink filled vacuoles, it is more difficult to count the number of microsphere filled vacuoles (especially if you use the larger sizes). Therefore, you might design an experiment in which you quantitate the percentage of Tetrahymena that phagocytose beads of a particular size (as opposed to counting the number of filled vacuoles after exposure to different bead sizes). Alternatively, you might try to count filled vacuoles. A basic protocol is presented below.

1. Add 40 µl of Tetrahymena to 40 µl of each size 0.08% Polybead dyed microsphere. (If you want to add 2 different colored microspheres at once, you should determine if you want to keep the total microsphere concentration constant or if you want to keep a single mirosphere concentration constant.) You will probably want to remove a 20 microliter sample immediately in order to assess a "zero time" control (see step 2). Calculate the concentration of the polystyrene beads after this combination.
2. At several time points (0, 15, 30 min. are suggested but you may vary this), REMOVE a 20 µl aliquot from the reaction microfuge tube of Tetrahymena/beads and, IN THE HOOD, put those aliquots in a clean, labeled microfuge tube containing 10 µl of 3% gluteraldehyde. Incubate for one minute then add 2 ul of 2.5M glycine. Incubate for one minute. This will stop phagocytosis by fixing the cells so you can study them later. If you would prefer to study your cells live, you will have to take your measurements very quickly to keep the timing relatively accurate. What is the effective concentration of the gluteraldehyde fixative?
3. Make a slide for each time point IN THE HOOD by placing 20 µl of each tube from step 4 on separate slides and cover each with a cover slip. Don’t make all your slides at the same time or they will dry out before you can count them.
4. Count or assess whatever you have decided to measure in as many cells as you can for each time point.
   
   

Don't worry that you may not be able to measure everything you would like to measure because of limited resources, equipment, and time available. Simplicity in your experimental design usually makes execution less difficult and the results more reproducible.

The following Reference Articles can be found on Sakai:
General Articles on Tetrahymena Phagocytosis
Gronlien, H.K., Berg, T., and Lovlie, A.M. (2002). In the polymorphic ciliate Tetrahymena vorax, the non-selective phagocytosis seen in microstomes changes to a highly selective process in macrostomes. J. Exp. Biol. 205, 2089-2097.

Jacobs, M.E., DeSouze L.V., Samaranayake, H., Pearlman, R.E., Sui, K.W.M., and Klobutcher, L.A. (2006). The Tetrahymena thermophila phagosome proteome. Eukaryotic Cell 5, 1990-2000.

McLaughlin, N.B., and Buhse, H.E. Jr. (2004). Localization by indirect immunofluorescence of tetrin, actin, and centrin to the oral apparatus and buccal cavity of the macrostomal form of Tetrahymena vorax. J Eukaryot. Microbiol. 51, 253-257.

Suhr-Jessen, P.B. and Orias, E. (1979). Mutants of Tetrahymena thermophila with temperature sensitive food vacuole formation, 1. Isolation and genetic characterization. Genetics 92, 1061-1077.

General Article on Phagocytosis:
Desjardines, M. and Griffiths, G. (2003). Phagocytosis: latex leads the way. Current Opinions in Cell Biology 15, 498-503. doi: 10.1016/S09555-0674(03)00083-8.

Article on Actin role in Motility and Phagocytosis:
Williams, N.E., Tsao, C., Bowen, J., Hehman, G.L., Williams, R.J., and Frankel, J. (2006). The actin gene ACT1 is required for phagocytosis, motiliy, and cell separation of Tetrahymena thermophila. Eurkaryotic Cell 5,555-567. doi: 10.1128/EC.5.3.555-567.2006

Articles on Cytochalasin B
Gavin, R.H. (1976). The oral apparatus of Tetrahymena pyriformis, strain WH-6. 11. Cytochalasin B inhibition of oral apparatus morphogenesis. J. Exp. Zool. 197, 59-64.

Gavin, R.H. (1976b). The oral apparatus of Tetrahymena pyriformis, strain WH-6 111. The binding of the 3H-cytochalasinB by the isolated oral apparatus. J. Exp. Zool. 197, 65-70.

Hoffman, E.K., Rasmussen, L., and Zeuthen, E. (1974). Cytochalasin-B: aspects of phagocytosis in nutrient uptake in Tetrahymena. J. Cell Set. 15, 403-406.

Nilsson, J.R.,. Ricketts, T.R., and Zeuthen, E. (1973). Effects of cytochalasin B on cell division and vacuole formation in Tetrahymena pyriformis. GL. Exptl Cell Res. 79, 456-459.

Articles on Colchicine
Kovacs, P. and Csaba, G., (2006). Effect of drugs affecting microtubular assembly on microtubules, phospholipid synthesis and physiological indices (signalling, growth, motility and phagocytosis)in Tetrahymena pyriformis. Cell Biochem Funct. 24, 419–429. DOI: 10.1027/cbf.1238

Stargell, L.A., Heruth, D.P., Gaertig, J., and Gorovsky, M.A. (1992). Drugs Affecting Microtubule Dynamics Increase cx-Tubulin mRNA Accumulation via Transcription in Tetrahymena thermophila. Molecular and Cell Biology 4, 1443-1450. DOI:0270-7306/92/041443-08$02.00/0

Article on DMSO
Nilsson, J.R., (1974). Effects of DMSO on vacuole formation contractile vacuole function, and nuclear division in Tetrahymena pyriformis GL. J. Cell Sci. 16, 39-47 .

Laboratory Cleanup

1. Place used micropipette tips and microcentrifuge tubes in autoclave bags on your bench.
2. Rotate the 4x objective lens into place.
3. The binocular head must be rotated into the storage position, to protect the ocular lenses from damage. Loosen the setscrew on the right, rotate the head 180°, then tighten the screw. Turn off the microscope light. Return the microscope to the cabinet under your bench with its plastic cover on.
4. Put all used microscope slides and cover slips in the glass disposal box.
5. Anything that was exposed to gluteraldehyde but NOT exposed to glycine should have been disposed of in the hood.

Assignments

Preparation for Science Writing Workshop in Lab 5

1. (10 points)In lab 5, you and your partner or group will give a 5 minute oral presentation showing your data and explaining how it addresses your experimental question and topic. Please prepare a few PowerPoint slides that include at least one figure and figure legend(s) summarizing the main points from your data. Although figure legends are not typically included in PowerPoint presentations (since this is the part that you explain verbally), you will include the legend this time so we can discuss effective figure design. Be sure and upload your presentation to the folder your instructor designates in the lab Sakai site before LAB5 begins so that you can access it on the computer in the presentation classroom. We will not be meeting in the lab for your presentations. Your instructor will let you know where to meet.
   
   
2. Read the Gronlien, et al. (2002) journal article again carefully. Now that you have done your own investigation of phagocytosis in Tetrahymena and created your own figures from your data, you are better equipped to think about and to discuss how scientific findings are presented. We will have a Science Writing Workshop after the presentations in LAB 5. Pay careful attention to the format of the different sections of the Gronlien paper. This time you will be reading the paper for style more than for content. The main purpose of our workshop is to help you understand the structure of scientific writing so that you will be prepared to write a partial scientific research paper of your own.