BISC110: 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. POTENTIAL INHIBITORS OF VACUOLE FORMATION IN TETRAHYMENA PHAGOCYTOSIS'

You will have the opportunity to test how either a cytoskeleton inhibitor or a phosphatase inhibitor affects vacuole formation in phagocytosis. The cytoskeleton, which consists of intermediate filaments, microtubules, and actin filaments, plays an important role in the structure and mobility of cells and cell components. You can test to see if actin and/or microtubules play a role in food vacuole formation in Tetrahymena.

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. Today in lab, you may choose to test whether 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. CAUTION: Wear gloves and exercise caution when handling the inhibitors because they are toxic.

Cytoskeleton Inhibitors
A. Cytochalasin B is an inhibitor of actin.
B. Colchicine is an inhibitor of microtubules.

Phosphatase Inhibitors
A. Phenylarsine oxide (PAO) is an inhibitor of tyrosine phosphatases.
B. Cantharadin is an inhibitor of serine/threonine phosphatases.

GENERAL PROTOCOL (can be modified)

Stock concentrations of inhibitors available to you:
PAO 300mM (50mg.ml) in DMSO diluent
Cantharadin 127 mM (25mg/ml) in acetone diluent
Cytochalasin B 14mM (6.7mg/ml) in DMSO diluent
Colchicine 25mM (10mg/ml) in water diluent

1. In a microfuge tube, make a 1:100 dilution of 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 1-10 min. (Note that you can omit this pre-incubation step altogether if your research indicates that would be a better to immediately start the test for inhibition of phagocytosis without pre-incubation.) What is the effective concentration of inhibitor in this important step?

To test for the effect of inhibitor on Tetrahymena phagocytosis:
3. Add 100 µl of 1% India ink. Recalculate the concentration of your inhibitor and India ink in this step. (Note that you are making another ~1:100 dilution of your inhibitor.)

4. At several time points (0, 1, 5, 10, 20 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 put that aliquot in a new, clean, labeled microfuge tube containing 10 µl of 3% gluteraldehyde. This will stop the reaction. What is the effective concentration of the gluteraldehyde fixative?

5. Make slides for each stopped reaction by placing 20 µl of each tube on separate slides and cover each with a cover slip. )Don’t make all your slides at once or they will dry out before you can count them.)

6. Count the number of filled vacuoles per cell in at least 20 cells for each time point (determined by how consistent data is in first 10).

Do the procedure again in exactly the same way but substitue 1 microliter of water for your inhibitor so you can compare the rate of vacuole filling and formation in the presence and absence of your inhibitor. If your inhibitor is diluted in something other than water, do you need to do a third experiment substituting 1 microliter of your diluent for the inhibitor? Why or why not?

This suggested protocol is one way to evaluate the effect of your inhibitor on the rate of formation and filling of vacuoles in phagocytosis, but you may discover from your research that there are other aspects or steps in the process of phagocytosis that might also be impacted by your inhibitor. Don't worry that you may not be able to measure everything you would like to measure next week because of limited resources, equipment, and time available. Simplicity in your experimental design usually makes execution less difficult, but you may alter this suggested protocol if your instructor approves.

You will need to use the research article 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?

Calculations Involved in Designing Your Experiment

Calculate how you will make a working dilution of inhibitor from the provided inhibitor stock with the goal of achieving an appropriate concentration of inhibitor in the Tetrahymena/inhibitor preincubation time (if you choose to have a preincubation) or during the phagocytic process (after you add the India ink but before adding the gluteraldegyde fixative). If you are using the suggested protocol and 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 India ink to the 1:100 Tetrahymena/inhibitor solution prepared in step 1, then the inhibitor is effectively diluted 1:201.

Reference Articles:

Articles on Inhibitors:
PAO
Massol P, Montcourrie P,Guillemot J, Chavrier P. (1998) FC receptor-mediated phagocytosis requires CDC42 and Rac1. EMBO.(17):21, 6219-6229.

Teixeira JE and Mann BJ, (2002) Entamoeba histolytica-Induced Dephosphorylation in Host Cells. Infection and Immunity, 70 (4):1816-1823. DOI: 10.1128/IAI.70.4.1816–1823.2002

Yanaga F, Asselin J, Schieven GL, Watson SP. (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 DC, Kou CA, Png KJ, Moore MAS, (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 FU, Mu¨ller T, Peter Nacke, Schmitz W, Vahlensieck U, aNeumann J.,(1998), Contractility and Inhibition of Protein Phosphatases by Cantharidin. Gen. Pharmac. 31, 5: 729–733.

SAMARI HR.,MØLLER MTN, HOLDEN L, ASMYHR T, SEGLEN PO. Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins. Biochem. J. (2005) 386, 237–244

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 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.

HOFFMANN EK, RASMUSSEN L., ZEUTHEN E, (1974) CYTOCHALASIN B: ASPECTS OF PHAGOCYTOSIS IN NUTRIENT UPTAKE IN TETRAH YMENA. J. Cell Set. 15, 403-406.

NILSSON JR,. RICKETTS TR, ZEUTHEN E (1973) Effects of cytochalasin B on cell divisionand vacuole formation in Tetrahymena pyriformis GL. Exptl Cell Res. 79: 456-459.

Colchicine
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

STARGELL LA, HERUTH DP, GAERTIG J. AND GOROVSKY MA, (1992) Drugs Affecting Microtubule Dynamics Increase cx-Tubulin mRNA Accumulation via Transcription in Tetrahymena thermophila. MOLECULAR AND CELLULAR BIOLOGY. 12: (4):1443-1450. DOI:0270-7306/92/041443-08$02.00/0

DMSO
J. R. NILSSON (1974) EFFECTS OF DMSO ON VACUOLE FORMATION, CONTRACTILE VACUOLE FUNCTION, AND NUCLEAR DIVISION IN TETRAHYMENAPYRIFORMIS GL. J. Cell Sci. 16, 39-47.

Laboratory Cleanup

1. Place used micropipette tips and microcentrifuge tubes in autoclave bags taped above 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 retighten 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 have the opportunity to give a 5 minute oral presentation of your data. Please prepare a PowerPoint presentation that includes a figure and figure legend summarizing your lab 3 data. Although figure legends are not typically included in PowerPoint presentations, you will include one this time so we can discuss effective figure design.

2. Read the journal article assigned by your instructor that will be discussed in the Science Writing Workshop next time. Pay careful attention to the format of the different sections of the paper. You and your partner may be assigned figure or other part of the paper to present informally to the class as part of the science writing workshop discussion we will have in lab 5. Don’t worry if you have trouble understanding every detail in the paper. The main purpose of this exercise is to help you understand the style of scientific writing and how data is presented efffectively in figures.