Cell Bio Exam 1 2022

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Johns Hopkins University *

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AS.020.306

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Biology

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Apr 3, 2024

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Cell Biology 2022 Exam 1 1 Exam Guidelines/Instructions Please read the following list of instructions and expectations relevant to this exam. • This exam is open-book which means you can use any resources except other people. The questions are designed to be answered based on content provided in the class or critical- thinking/application of that content. Outside sources should be evaluated carefully. • Collaborations, conversations, or any other type of communication or sharing of information with any other individuals about the exam or relevant topics during the entire exam window is entirely prohibited. Any electronic communication, posting, or requesting of any information related to the exams is entirely prohibited during the entire exam time. It is imperative for the integrity of our community that each of you strictly adheres to these ethical exam expectations. If you become aware of any violations of these expectations at any time, you are obligated to inform Dr. Tifft. Any violations will be handled appropriately. • All answers must be your own work and consist of your own words and images (avoid any form of plagiarism …including copying directly from course materials or other study materials you may have prepared ahead of this exam). • You can type or write your answers on the PDF. Please do not alter the pagination or position of questions and answer spaces provided. Please make sure the file you submit has your answers on it. • The recommended answer length provided for some questions is a recommendation and should be interpreted as guidance (not a strict limit). For answers with a recommended answer length listed, please limit the answer in the space provided to the number of words indicated. If you want to add more words, please make sure the space provided contains the most important part of your answer and write any additional information in space provided in at the end of this exam. We can consult the extra information if needed. • Use the space provided in the last question on the exam to write any notes about your answers that you would like to include. • If you have questions, find any problems, or need clarification during the exam, please email Dr. Tifft ( tifft@jhu.edu ). This is the only exception to communicating with other people about the exam during the exam time. Learning assistants are not available to answer questions or provide guidance during the exam and should not be contacted. Please type/write your name here as an indication that you have read, understand, and agree to fully comply with the instructions and expectations listed above. ________________________

Cell Biology 2022 Exam 1 2 Western blot analysis and imaging 1. You study the expression of Protein A and Protein B using microscopy and Western blot analysis. You treated your sample with BME before running on SDS-PAGE for Western blot. 1A. Based on the results shown above, what can you tell about the properties of Protein A? Select all that apply. 3 points a) A fraction of Protein A interacts with protein B b) Protein A oligomerizes with other molecules of Protein A in the presence of Protein B c) A fraction of Protein A is post-translationally modified when co-expressed with protein B d) Protein A and Protein B co-localize in cells e) In lane 2, the amount of protein A is the same as the amount of protein in the loading control. Answer: _____________ 1B. What is an advantage of the general type of microscopy (white-light, fluorescence, or electron) shown in the images above? Select all that apply. 3 points a) The contrast is higher than white-light microscopy b) The contrast is higher than fluorescence microscopy c) The contrast is higher than electron microscopy d) The resolution is higher than white-light microscopy (lower limit of resolution) e) The resolution is higher than fluorescence microscopy (lower limit of resolution) f) The resolution is higher than electron microscopy (lower limit of resolution) Answer: _____________

Cell Biology 2022 Exam 1 3 CYTOSKELETON and MOLECULAR MOTORS 2. You are studying treadmilling of F-actin at steady state. Explain what occurs during F- actin treadmilling. Be sure to address the critical concentration (Cc) of the minus end (Cc-), plus end (Cc+), and the filament (Cc filament ), the on rate and off rates of subunits for the filament, and the concentration of G-actin ATP . (Recommended answer length <150 words) 6 points 3A. You attach individual myosin II motors (not bundles) on a coverslip. You add fluorescently-labeled stabilized actin filaments. Based on the images (taken every 5 seconds), what is the polarity of the left and the right end of the F-actin filament marked with the letter A? 5 points a) Right is – end, left is + end b) Right is + end, left is - end Answer: ______________ Explain your reasoning. Make sure to thoroughly address information about the experiment and proteins that lead to your conclusion. (Recommended answer length <100 words

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Cell Biology 2022 Exam 1 4 3B. You continue to image the dynamic behavior of these filaments for two more hours. You notice that the length of these filaments has not changed much during this time. You note that you generated F-actin filaments in a test tube using a whole cell extract that contained large amounts of actin ATP monomers and actin regulatory proteins. Which regulatory proteins could produce this effect on F actin length? Select the best answer. 2 points a) F-actin stabilizing proteins (such as tropomyosin) b) end capping proteins c) proteins that sequester G-actin ATP d) proteins that slow down ATP hydrolysis Answer:_____________ 3C. Discuss how the type of proteins you chose as the answer above influence the on rate (polymerization) and off rate (depolymerization) of F-actin and how the proteins change the observed critical concentration of the entire filament. (Recommended answer length <100 words) 4 points 4. You start with a solution of purified G-Actin ATP and allow actin filaments to form and reach steady-state. The Cc of the – end is 11 µM and Cc of the + end is 3 µM. 4A. If you raise the concentration of G-Actin ATP to 16 µM and observe a few minutes afterwards what would you expect to happen to actin filaments? Select the best answer. 2 points a) growth at plus and minus ends b) plus ends grow and minus ends shrink c) plus ends shrink and minus ends grow d) shrinking at plus and minus ends e) no growth or shrinking Answer: _______________

Cell Biology 2022 Exam 1 5 4B. If you lower the concentration of G-Actin ATP to 7 µM and observe a few minutes afterwards what would you expect to happen to actin filaments? Select the best answer. 2 points a) growth at plus and minus ends b) plus ends grow and minus ends shrink c) plus ends shrink and minus ends grow d) shrinking at plus and minus ends e) no growth or shrinking Answer: _______________ 4C. If you lower the concentration of G-Actin ATP to 1.5 µM and observe a few minutes afterwards what would you expect to happen to actin filaments? Select the best answer. 2 points a) growth at plus and minus ends b) plus ends grow and minus ends shrink c) plus ends shrink and minus ends grow d) shrinking at plus and minus ends e) no growth or shrinking Answer: _______________ 4D. If you raise the concentration of G-Actin ATP to 16 µM and add 5 µM activated Arp2/3 complex, describe what you would expect to observe after a few minutes. Be sure to describe the growth/shrinking at ends of filaments and any other aspects of filament dynamics and/or organization. Contrast this situation with the condition described in 4A (without Arp2/3). (Recommended answer length <100 words) 5 points

Cell Biology 2022 Exam 1 6 5. You decided to investigate microtubule dynamics using microtubules grown in a test tube and Western blot analysis. You investigate four (4) conditions listed in the table. Assume the critical concentration (Cc) of the filament is 5 μ M. Sample / lane Starting concentration tubulin GTP heterodimers Additional Components added 1 16 µM None 2 16 µM Axonemal dynein +ATP 3 16 µM Axonemal dynein +ATP + linking protein 4 10 µM None You allow all four samples to reach steady state, then isolate microtubules with any filament-associated proteins (discarding the proteins that are not associated with filaments including free tubulin GTP sununits). You add BME to all samples and load them on SDS-PAGE gel. After transfer you western blot for β-tubulin using a fluorescently- labeled antibody. Which image best shows what you anticipate to observe? 6 points Answer: ____________ Explain your reasoning. Be sure to address relevant aspects of microtubules and western blots to explain the results observed. (Recommended answer length <100 words)

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Cell Biology 2022 Exam 1 7 CELL MIGRATION 6. Your lab studies skeletal muscle contraction. Using genome editing, you create a mouse that express a myosin II that can bind ADP but cannot bind ATP. How would you predict this mutant to affect myosin II movement, myosin: actin interaction (binding) and sarcomere contractility in these mice? Explain your reasoning. Be sure to fully address the function of myosin. (Recommended answer length <100 words) 5 points 7. You decide to study cell migration in a dish coated with fibronectin. Using genome editing, you create cells that express constitutively activated Cdc42GTPase OR integrins that cannot bind to actin cytoskeleton. The proteins are expressed throughout the entire cell and the cells only express the mutant versions of the proteins. How would you expect migrating cells to be affected? Select the best answer for each type of mutant protein. 5 points Constitutively activated Cdc42GTPase _____________ Integrins that cannot bind to actin cytoskeleton _____________ Answer Choices: a) Would not form filopodia and thus would not migrate b) Would continuously form stress fibers throughout the cell c) Would form filopodia throughout the cell but would not migrate in one direction d) Would not form lamellipodia and would move more slowly e) Would not form focal adhesions, would not attach to fibronectin, no traction f) Would move more quickly as the retraction would be easier and thus faster END OF EXAM QUESTIONS

Cell Biology 2022 Exam 1 8 Please use this optional space to tell us anything you want us to know about the questions or your answers while we are grading the exam. You can provide explanations, state assumptions, explain confusions, or describe any concerns about specific questions. We can consult any information provided here when grading or regrading the exam if necessary.

Cell Bio Exam 1 2022

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