I need to write a lab report including Abstract, Introduction, Material and Methods, Results and Discussion
Roles of Actin and Microtubules in Cellular Motility Cell Biology Fall 2020 In this lab, you will study the role of two filaments of the cytoskeleton, microtubules and actin microfilaments, in motility, phagocytosis, and food vacuole formation using the oragnism Tetrahymena. Tetrahymena are a ciliated protozoa (a group of unicellular eukaryotes). Tetrahymena feed by phagocytosis; a form of endocytosis that allows them to ingest bacteria or other small pieces of food from the environment. Food ingested by phagocytosis ends up in intracellular compartments called food vacuoles. Phagocytosis The ability of cells to change their shape and engulf particles during phagocytosis requires the cytoskeleton. You will use cytochalasin B, an inhibitor of actin microfilaments, and colchicine, an inhibitor of microtubules, to determine whether actin microfilaments or microtubules are more important for the process of phagocytosis. By treating tetrahymena with these inhibitors you will disrupt actin or microtubule function in those cells. You can then measure whether phagocytosis is blocked when you inhibit those specific cytoskeletal proteins. Phagocytosis in Tetrahymena can be studied by allowing cells to swim and feed in India ink. Tetrahymena will ingest the ink while feeding, and the ink will end up in food vacuoles. This will show up clearly as black spots in the cell. Cells that have been feeding by phagocytosis in india ink will have dark ink-filled spots (food vacuo1es) Q1. What is the purpose of the experiment? Q2. How will you test it (in what organism, with what tools?) With your group, go through each step of the procedure. Discuss why you do each step, and what you expect to see. Experiment 1: Preliminary observations Part 1: Objective: Observation of Tetrahymena and formation of food vacuoles. (1)Set up your microscope. (2)You have been provided with a culture of Tetrahymena grown in proteose peptone (food). Remove a small sample (20µl) of Tetrahymena suspension and place on a clean microscope slide. Place under a coverslip and observe under the 40X objective. You see the following: (3)Using a micropipettor, measure out 50 µl of Tetrahymena suspension into a clean 1.5 ml microcentrifuge tube. Add an equal volume (50 µl) of 1% India ink. Start the timer for 10 minutes (5)Ten (10) min. after adding the ink, remove 20 µl of cells in ink into a clean 1.5 ml microcentrifuge tube (5)Observing the cells under (40X) and look for black food vacuoles in the Tetrahymena you see the following:. (a) Tetrahymena before ink. (b) Tetrahymena after being fed 1% ink for 10 minutes: Q 3. Why did you put the tetrahymena in ink? Q4. What is the reason for the difference you see in the images of cells before and after adding ink? Q5. What is the purpose of this part of the experiment? Part 2: (1)Measure out 100µl of Tetrahymena suspension into each of three clean test tubes ;labelled A, B and C. (2)- Into the first tube (A) add 25 µl of cytochalasin B (final concentration of 100 µg/ml) - Into the second tube (B) add 25 µl of colchicine (final concentration of 5mg/ml) - The third tube(C) is a control - what do you think you should add to this tube and why? (3)Let the cells sit in drugs for 10 min at room temperature to allow the inhibitors to inhibit their targets. While you are waiting, label twelve tubes as follows · A0, B0, C0 · A15 B15, C15 · A30, B30, C30 (A,B,C = treatment condition (see step 1 and 2), 0, 10, 20 etc. = time after adding ink 10 minutes…20 minutes… ) (4)After 10 min, add 125ul of 1% India ink to each of the three tubes (A, B and C). · Start your timer now. · Immediately take 20ul of cells from each condition (A, B and C) and add it to the appropriately labelled tube (A0, B0, C0). · Specifically, take 20ul of cells from Tube A and place it into the tube labelled A0. Repeat the same for tubes B (20 uL from B into B0) and C (20 ul from C into C0)- This is the time 0 sample- the cells have been feeding for 0 minutes. (6)Repeat this sampling every ten minutes: At 15 minutes take 20 uL from tube A and add to tube A15. Repeat the same for tubes B (20 uL into B15) and C (20 ul into C15)- This is the 15 minute time sample- the cells have been feeding for 10 minutes. Repeat this at 30 minutes. Use diagram below to show how you will carry out this part of the experiment. 15 min 15 min 15 min 15 min Q6. Why do you have tube C? What were the drugs added to tubes A and B used to test? Q7. Based on what you know from discussion of cytoskeletal proteins, what is your prediction for the results of his experiment (will wither drug affect phagocytosis or the formations of vacuoles)? Why? Results: You have cells that have been feeding ink by phagocytosis for 4 different lengths of time (Cells feeding for 0, 15, and 30 minutes) with three different treatment conditions (cytochalasin(A), colchicine (B), and control (no drug, C)). Twelve total samples. Count how many vacuoles have formed in the tetrahymena for each of the twelve time samples. Count vacuoles in 10 different cells and calculate the average for each condition. Plot the average (mean) number of vacuoles for each cell against time for each treatment. Use this data to establish: What were the effects of cytochalasin B and colchicine? Are microfilaments, microtubules, or both required for phagocytosis and vacuole formation in Tetrahymena? Make sure to explain this in your lab report. For each condition (image A0, B0, C0 etc) count how many vacuoles are in each tetrahymena. Analyze 10 tetrahymena in each. Calculate the average for each condition. Example:A10 –counting 10 cells -they have 2 3 2 4 5 6 5 3 4 6 vacuoles each. Sum =40 Average=40/10=430 15 Acknowledgements:This protocol was modified from M. Wilson and M. Olney (St. Martin’s University based on : Bozzone, D.M. (2000). Investigating phagocytosis in Tetrahymena: An experimental system suitable for introductory and advanced instruction. The American Biology Teacher 62: 136-139. PowerPoint Presentation A0 B0 C0 A15 B15 C15 A30 B30 C30