BISC110/S11: 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 test an experiment you and your partner design. Each group of two students will focus on one inhibitor, and no more than two student groups can work on any one inhibitor.

I. Factors Affecting Tetrahymena Phagocytosis
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. 

Because protein function is dependent on protein shape and charge, many biochemical pathways add a phosphate group to a protein as an effective way to regulate protein function. The addition of a phosphate group is termed phosphorylation, and an enzyme that adds a phosphate group to an amino acid within a protein is called a protein kinase. In contrast, protein phosphatases are enzymes that dephosphorylate (remove a phosphate group from) certain amino acids within proteins. Depending on the protein, phosphorylation can either activate or inhibit protein function, and it plays an important role in many cell signaling events that occur in the cell. You may choose to test whether one of two protein phosphatases are important for food vacuole formation in Tetrahymena.

All of the reagents that you used previously will be available again for this experiment. In addition, you will be able to use one of the following inhibitors of the elements whose role you want to test. CAUTION: Wear gloves and exercise caution when handling the inhibitors because they are toxic.

Main Topic Choices: Is phagocytosis in Tetrahymena pyriformis selective or non-selective for particle type or size?

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? (Use actin inhibitor compared to a reaction without inhibitor Cytochalasin B, inhibitor of actin)

'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''? '''Use tubulin inhibitor compared to phagocytosis without inhibitor: Colchicine or Nocodazole, both inhibitors of tubulin

'What role does tyrosine phosphatase or threonine phosphatase play in some aspect of phagocytosis (either engulfment, vacuole formation, filling, movement or vesicle fusion) in Tetrahymena pyriformis''? '''Use either of two phosphatase inhibitors (inhibits various protein activation or deactivation events):  Phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases or Cantharadin, an inhibitor of threonine phosphatases

Stock concentrations of reagents available to you: PAO 300mM (50mg/ml) in DMSO Cantharadin 127 mM (25mg/ml) in acetone Cytochalasin B 14mM (6.7mg/ml) in DMSO<BR> Colchicine 25mM (10mg/ml) in water<BR> Nocodazole 2mg/ml (7mM) in DMSO<BR> 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. <BR> Polybeads- blue dyed polystrene sizes available: <BR> 0.2µm (conc.= 5.68 x 1012 particles/ml Coefficient of Variation (CV) 5% <BR> 0.5µm (conc =3.64 x 1011 particles/ml Coefficient of Variation (CV) 3%) <BR> 1.0µm (conc= 4.55 x 1010 particles/ml Coefficient of Variation (CV) 3%) <BR> 3.0µm (Conc= 1.68 x 109 particles/ml Coefficient of Variation (CV) 5% )<BR> Polybeads packaged as 2.5% aqueous suspension (water) <BR><BR>

ONE POSSIBLE PROTOCOL (can be modified)<BR><BR>
 * 1) In a microfuge tube, make a 1:100 dilution of 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 viability of your cells if in significant concentration.) Include an appropriate control for your experiment to make sure that your inhibitor's diluent is not affecting vacuole formation?
 * 2) Incubate for 0-10 min. if you think your inhibitor needs time to affect the element (actin, tubulin, cilia, 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) Start here if you are not testing a drug inhibitor of protein function or a cytoskeleton element in Tetrahymena phagocytosis: Add 100 µl of 1% India ink or whatever "food source" at whatever concentration you think appropriate to the inhibitor+ Tetrahymena mixture prepared in step 1 OR (if you are not using inhibitor) to 100 μL of  Tetrahymena in a microfuge tube. This addition starts phagocytosis so just before you add the food to start phagocytosis is considered your "zero time point". You will probably want to remove a 20 microliter sample immediately in order to assess a "zero time" control. Calculate the concentration of your inhibitor and India ink or other food after this combination . (Note that you are making another ~1:100 dilution of your inhibitor in this step.
 * 4) At several time points (0, 5, 10 min. are suggested but you may vary this), REMOVE a 20 µl aliquot from the reaction microfuge tube of Tetrahymena/inhibitor/ink or other food and the control tube of Tetrahymena/ diluent /food and put those aliquots in a new, clean, labeled microfuge tube containing 10 µl of 3% gluteraldehyde, if you want to study your cells fixed. You have stopped phagocytosis by killing 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?
 * 5) Make a slide for each time point by placing 20 µl of each tube 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.
 * 6) Count or assess whatever you have decided to measure in as many cells as you can for each time point.

Do the procedure again in exactly the same way but substitue 1 μL of water for your inhibitor so you can compare your measurements in the presence and absence of your variable. If you are using a drug inhibitor that is diluted in something other than water, do will need to do another control substituting 1 microliter of the diluent of the drug (DMSO or acetone)? Why do you need this control?<BR><BR>

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.<BR><BR>

You will need to use the research article or other information provided by your instructor to find a appropriate concentration of inhibitor to test in your experiment. Once you have that target final concentration in mind you will need to figure out a working dilution to make of the provided stock solution of inhibitor. What will you use for diluent? The directions below may be helpful in accomplishing that task.<BR><BR>

Calculations Involved in Designing Your Experiment<BR><BR> Calculate how you will make a working dilution of an inhibitor or "food" from the provided stock reagent with the goal of achieving an appropriate concentration in the crucial part of the experiment. This time might be the Tetrahymena/ inhibitor preincubation time (if you choose to have a preincubation) or it could be during the phagocytic process (after you add the India ink but before adding the gluteraldegyde fixative)if you don't have a preincubation period. If you are using the suggested protocol with a preincubation, please remember that the working dilution of inhibitor that you make from the stock must be 100x stronger than final concentration you desire because you are diluting your inhibitor 1:100 in step 1. If you don't have a pre-incubation period and immediately add the 1% India ink or other food to the 1:100 Tetrahymena/inhibitor solution prepared in step 1, then the food or drug inhibitor is effectively diluted 1:200 from the stock. <BR><BR> Reference Articles: General Articles on Tetrahymena Phagocytosis<BR> Gronlien, H.K., Berg, T., 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.<BR><BR>

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

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.<BR><BR>

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.<BR><BR>

General Article on Phagocytosis:<BR> 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. <BR><BR>

Article on Actin role in Motility and Phagocytosis:<BR> Williams, N.E., Tsao, C., Bowen, J., Hehman, G.L., Williams, R.J., 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<BR><BR>

Articles on Inhibitors: PAO Massol, P., Montcourrie, P. ,Guillemot, J., Chavrier, P. (1998). FC receptor-mediated phagocytosis requires CDC42 and Rac1. EMBO. 17, 6219-6229.

Teixeira, J.E. and Mann, B.J., (2002) Entamoeba histolytica-induced dephosphorylation in host cells. Infection and Immunity, 70, 1816-1823. DOI: 10.1128/IAI.70.4.1816–1823.2002

Yanaga, F., Asselin, J., Schieven, G.L., Watson, S.P. (1995). Phenylarsine oxide inhibits tyrosine phosphorylation of phospholipase Cy2 in human platelets and phospholipase Cyl in NIH-3T3 fibroblasts. FEBS Letters, 368, 377-380.

Cantharadin Dorn, D.C., Kou, C.A., Png, K.J., Moore, M.A.S., (2009). The effect of cantharidins on leukemic stem cells. Int. J. Cancer: 124, 2186–2199.

Knapp, J., Boknı,´k. P., Huke, S., Gombosova, I.´, Linck, B., Lu¨ss, H., Mu¨ller, F.U., Mu¨ller, T., Nacke, P., Schmitz, W., Vahlensieck, U., aNeumann, J.,(1998). Contractility and Inhibition of Protein Phosphatases by Cantharidin. Gen. Pharmac. 31, 729–733.

Samari, H.R..,MØller, M.T.N., Holden, L., Asmyhr, T., Seglen, P.O. (2005). Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins. Biochem. J. 386, 237–244. <BR><BR>

PAO + Cantharadin Kovacs P and Pinter M., Effects of phosphoprotein phosphatase inhibitors (phenylarsine oxide and cantharidin) on Tetrahymena. (2001) Cell Biochemistry and Function, 19, 197-205.

Cytochalasin B<BR> Gavin RH (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 RH, (1976 -2) 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., Zeuthen, E., (1974) Cytochalasin-B: aspects of phagocytosis in nutrient uptake in Tetrahymena. J. Cell Set. 15, 403-406.<BR><BR>

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

Colchicine<BR> Kova´cs, 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<BR><BR>

Stargell, L.A., Heruth, D.P., Gaertig, J., 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<BR><BR>

DMSO<BR> 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 .<BR><BR>

Laboratory Cleanup

 * 1) Place used micropipette tips and microcentrifuge tubes in autoclave bags on your bench.
 * 2) Clean the oil immersion objective lens (100x) of your microscope using only lens tissue (NOT Kimwipes®). Check the 40x objective lens as well.  Sometimes it gets inadvertently contaminated with immersion oil.
 * 3) Rotate the 4x objective lens into place.
 * 4) 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.
 * 5) Put all used microscope slides and cover slips in the glass disposal box.

Assignments
Preparation for Science Writing Workshop in Lab 5
 * 1) In lab 5, you and your partner will give a 5 minute oral presentation showing your data and explaining how it addresses your experimental question or topic. Please prepare a few PowerPoint slides that include a figure and figure legend summarizing your lab 3 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 our First Class lab conference 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 Gronlein, 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 equiped 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 report of your own.<BR><BR>

Links To Other Labs in this Series
Lab 1: Boot Camp Lab 2: Scientific Investigation in Tetrahymena Lab 3: Dilutions & Scientific Investigation in Tetrahymena Lab 4: Variable Testing in Tetrahymena Lab 5: Scientific Writing & Effective Figure Design