Copy of Lab 3 Post-lab_FA22

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Post-lab Assignment LAB 3: Cells Bio 1AL FALL 2022 Last Name _______Kim_________ First Name __________Juliana___________Lab Section #______223_______ Lab 3 Post-Lab 1) (1 pt) In the field of view circle below, draw an Amoeba at 200X total magnification (using the 20X objective). a) Label the following structures: pseudopods, food vesicles (and contractile vacuole if observed ) . b) Include a scale bar with clearly labeled length and units. Note m does not equal M. µ µ Drawing instructions: If you hand-drew the drawings, you can take a picture of the drawings and insert the image below. Total Magnification _____200X______ 3-1

Post-lab Assignment LAB 3: Cells Bio 1AL FALL 2022 2) (3 pt) In the left field of view circle below, draw a Paramecium cell and a few surrounding yeast cells. a) Label the following structures: cilia, contractile vacuole, yeast cell and oral groove b) Include a scale bar with clearly labeled length and units. c) Use the reticle calibration from Lab 2 to determine the length of the Paramecium , and the diameter of the indicated yeast cell. Include appropriate units in your answers in Table 1 below. On the right field of view circle below, draw a stained cheek cell using this image. d) Label the following structures: nucleus, cell membrane, cytoplasm e) Include a scale bar with clearly labeled length and units. Note m does not equal M. µ µ f) Use the reticle calibration from Lab 2 to determine the diameter (widest part) of the cheek cell, and the diameter of the nucleus. Include appropriate units in your answers in Table 1 below. Drawing instructions: If you hand-drew the drawings, you can take a picture of the drawings and insert the image below. Total Magnification ______400X?_______ Total Magnification _______1000X______ Table 1. Calculated Dimensions of Cellular Structures (1 pt) Paramecium Cheek Cell Total magnification: 400X Total magnification: 1000X Length (long axis): 220 m µ Cheek cell diameter: 50 m µ Yeast cell diameter: 21 m µ Nucleus diameter: 7 m µ 3-2

Post-lab Assignment LAB 3: Cells Bio 1AL FALL 2022 3) Follow the Lab 3 procedures to measure rates of cytoplasmic streaming in Nitella . Record your streaming rates below and on the class data sheet - use two decimals for your rates. For the class data sheet, record your streaming rate in the corresponding green cells in the class data sheet by station number. Baseline rate (pre-FCCP) Rate After FCCP Treatment Experiment 1 5.34 s 10.26 s Experiment 2 6.13 s 9.84 s 4a) Once all streaming times and streaming rates have been entered for all lab station groups in your lab section, download your class datasheet as an Excel File ( File > Download > Microsoft Excel ). 4b) In Excel, calculate the average, standard deviation, and standard error of the mean (See Excel Graphing Resources page) for the cytoplasmic streaming rates before (baseline) and after FCCP addition (cells highlighted blue ), using your lab section’s class data. Copy the values for the average streaming rates and SEM into Table 2 below. 4c) Calculate the percentage change between the two means and enter into Table 2. See the Comparing Two Means page in bCourses. 4d) Run a Correlated (Paired) Sample t-test in VassarStats comparing the average baseline cytoplasmic streaming rate to the average streaming rate after FCCP addition. See the Paired/Correlated Sample t-test page in bCourses. Use the one-tailed p-value , since we are predicting that FCCP will decrease cytoplasmic streaming rate (i.e. we are predicting an effect in one specific direction). Record the relevant values in Table 2 below. Table 2. Effect of FCCP on cytoplasmic streaming rate in Nitella Baseline After FCCP Mean streaming rate ± SEM (μm/sec) (0.5 pt) 47.393 ± 4.502 seconds 21.197 ± 3.402 seconds Percentage change (%) (0.5 pt) 55.27% Paired t-test results (0.5 pt) t = 6.84 , df = 21 , p (1-tailed) < 0.0001 5a) (0.5 pt) What is your null hypothesis for the t-test? Our null hypothesis is that the difference between the average cytoplasmic streaming rate before and after the addition of FCCP is zero. 5b) (0.5 pt) Does FCCP addition significantly decrease cytoplasmic streaming rate? Circle/highlight: YES / NO 3-3

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Post-lab Assignment LAB 3: Cells Bio 1AL FALL 2022 6) (1.5 pt) Using Excel, graph the average cytoplasmic streaming rate before (baseline) and after FCCP addition from the class data. Use a column graph in Excel, and a dd custom error bars, using your calculated SEM values. Save the graph in Excel as an image (Ctrl Click on the graph > Save as Picture or take a screenshot) and paste the image below. 7) (0.5 pt) We are missing a DMSO-only control experiment in our experimental design. In a few sentences, briefly describe the key steps of this control experiment. We want to see if DMSO was not responsible for the slower rate of cytoplasmic streaming, so we need to create a wet mount of Nitella and again use a camera to time how long it takes for a vesicle to travel 200 m. After we get that time, we add 100 L of DMSO to the edge µ µ of the cover glass slowly, wait 12 minutes, and then measure the streaming time again to get the DMSO-only streaming rate. 8a) (0.25 pt) How did the cytoplasmic streaming rate in Elodea compare to in Nitella? The streaming rate in Elodea was: Circle/highlight one: FASTER SLOWER 8b) (0.25 pt) Briefly discuss one factor that may cause a different streaming rate in Elodea . (1-2 sentences) One factor that could have caused a different streaming rate in Elodea is the difference in cell size or plant size, with Nitella having a larger cell size and leaf size in general, correlating with a higher cytoplasmic streaming velocity. 3-4

Post-lab Assignment LAB 3: Cells Bio 1AL FALL 2022 9) The next question refers to this paper that was discussed in Lecture 3.. The abstract and Figure 4 are shown below. 9a) (0.5 pt) Let’s start by only looking at the bars labeled “N” in Figure 4, which are the results of experiments using the normal myosin purified directly from scallops. Describe what you can conclude from comparing only the bars labeled “N” on the left side (no Ca 2+ ) and right side (0.1 mM Ca 2+ ) of Figure 4. From just looking at the bars labeled “N,” we can conclude that calcium does play an essential role in intracellular motility, as the motility of normal scallop myosin goes from almost zero with no calcium in the cytoplasm to over 2 m/s when 0.1mM of calcium was introduced in the cytoplasm. µ 9b) (0.5 pt) Now look only at the right half of Figure 4. Compare only the “N” bar (regulatory light chain present) to the “D” bar (no regulatory light chain present) . What does this tell us about the role of the regulatory light chain? Regulatory light chains in these cells are primarily responsible for controlling the movement of myosin toward actin filaments, and this is evident because with the “D” bar, it shows a very low velocity of streaming whereas with the regulatory light chain present, the motility velocity is higher. 3-5

Post-lab Assignment LAB 3: Cells Bio 1AL FALL 2022 10. An in vitro system was used to study myosin interactions from different species, in particular with actin bundles isolated and purified from Nitella . Vale and associates manipulated levels of calcium to study its role in regulating myosin activity. They also measured motility rates.. Using an in vitro system they were able to show motility rates varied with calcium levels and that myosin isolated from different species had different velocities. Table 1 illustrates the motility rates with and without calcium (two far right columns). You decide to see if the difference in velocities between your motility measurements and Vale’s measurements stem from differences in the myosin motor from Nitella you observed versus the myosin motor from Aequipecten and Placopecten that Vale used. a) (0.5 pt) Describe an experiment using Vale’s in vitro system to test this question. Using a petri dish and yeast cells, we can first isolate the actin filaments and purify them from the cells. We can also use the in vitro system that Vale utilized to place the yeast cells in differing levels of calcium environments to see the proliferation of motility rates varying along with comparisons of regulating myosin activity. b) (0.5 pt) Controls typically are required. A negative control would exclude myosin from the analysis. Describe an example of a positive control. A positive control could be to use differing levels of calcium environments with no manipulation of the myosin so that we will see a response that indicates our reagents work in the first place. 3-6

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