Cell and Molecular Biology: Problem Set 3 Guide

Cell and Molecular Biology (BIOL 4707) Problem Set 3 (50 POINTS TOTAL) This assignment, covering material from module 3, is due on Wednesday, February 14 th at 11:59 PM to be submitted via Canvas.  Verify that the submission was successful  Note: The space given per question is not based on the expected size of the answer. Be sure to answer all questions asked  Once the deadline has passed, 10% of the total assigned score will be deducted for each day it is late You should feel free (and indeed I encourage you) to discuss the problems in study groups with your classmates, but the final answers you submit must be your own: they must be written by you in your own words and reflect your own thinking. Please read the academic integrity policy of the course.

1. (4 points) The orientation of the  -tubulin dimer in a microtubule was determined in several ways. GTP-coated fluorescent beads, for example, were found to bind exclusively at the plus ends of microtubules. By contrast, gold beads coated with antibodies specific for a peptide of  -tubulin bound exclusively at the minus end. Which tubulin subunit,  or  , is at which end (2 points, + or -) ? Explain your reasoning (2 points, 1-2 sentences) . The B-tubulin subunit is located at the plus end, while the alpha-tubulin subunit is located at the minus end. ADD REASONING.

2. (6 points) The movements of single motor-protein molecules can be analyzed directly. Using polarized laser light, it is possible to create interference patterns that exert a centrally directed force, ranging from zero at the center to a few piconewtons at the periphery (about 200 nm from the center). Individual molecules that enter the interference pattern are rap- idly pushed to the center, allowing them to be captured and moved at the experimenter’s discretion. Using such “optical tweezers,” single kinesin molecules can be positioned on a microtubule that is fixed to a coverslip. Although a single kinesin molecule cannot be seen optically, it can be tagged with a silica bead and tracked indirectly by following the bead (Shown below) . In the absence of ATP, the kinesin molecule remains at the center of the interference pattern, but with ATP it moves toward the plus end of the microtubule. As kinesin moves along the microtubule, it encounters the force of the interference pattern, which simulates the load kinesin carries during its actual function in the cell. Moreover, the pressure against the silica bead counters the effects of Brownian (thermal) motion, so that the position of the bead more accurately reflects the position of the kinesin molecule on the microtubule. Traces of the movements of one kinesin molecule along a microtubule is shown below . a. (2 points) As shown in panel B, all movement of kinesin is in one direction (toward the plus end of the microtubule). What supplies the free energy needed to ensure a unidirectional movement along the microtubule (1 point, 1 word) ? Explain your reasoning (1 point, 1 sentence) ATP. The hydrolysis of ATP provides the free energy for the unidirectional movements of kinesin on the microtubule. b. (2 points) What is the length of each step that a kinesin takes as it moves along a microtubule (1 point, give approximate length) ? Show you reasoning (1 point, math or 1 sentence) . Approximate length: 8 nm. From the given figure, it can be inferred that the kinesin moves about 80 nm in 9 seconds, this gives an average of 80/9 = 8.89 nm/second. FIX REASONING. Figure. Movement of kinesin along a microtubule. (a) experimental set-up, with kinesin linked to a silica bead, moving along a microtubule. (B) position of kinesin (as visualized by the position of the silica bead) relative to the center of the interference pattern, as a function of time of movement along the microtubule. The jagged nature of the trace results from Brownian motion of the bead.

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c. (2 points) From other studies it is known that kinesin has two globular domains that can each bind to β-tubulin, and that kinesin moves along a single protofilament in a microtubule. In each protofilament, the β-tubulin subunit repeats at 8-nm intervals. Given the step length and the interval between β-tubulin subunits, how do you suppose a kinesin molecule moves along a microtubule (2 points, 1-2 sentences) ? Out of the two domains of kinesin that binds β-tubulin, one domain is anchored while the other swings towards the subsequent tubulin binding site. 3. (4 points) Cytochalasin B interferes with the assembly of actin filaments. In the classic experiment that defined its mechanism, short lengths of actin filaments were decorated with myosin heads and then mixed with actin subunits in the presence or absence of cytochalasin B. Assembly of actin filaments was measured by assaying the viscosity of the solution (see the figure Part A) and by examining samples by electron microscopy (Part B). The decorated actin filaments present before the addition of actin monomers are shown at the top of each set of three. Filaments present after increasing times of incubation with actin monomers (red circles) are shown below. Explain how this data supports the conclusion that Cytochalasin B functions as a plus end cap (4 points, 1-2 sentences) . 4. (9 points) The following graph shows the elongation rate at the plus and minus ends of actin filaments as a function of actin subunit concentration. Based on your knowledge of actin filaments, circle the line that represents the plus end (1 point, circle line) . For each concentration (A to E), state whether the filament is growing, shrinking, steady-state treadmilling (5 points, fill in table) ? Explain your reasoning (3 points, 1-2 sentences) . Letter Growing, Shrinking, or Treadmilling? A B C D E

5. (9 points) You isolate some muscle fibers to examine what regulates muscle contraction. When you bathe the muscle fibers in a solution containing ATP and Ca 2+ , you see muscle contraction (table below) . Ca 2+ is necessary, as solutions containing ATP alone or nothing do not stimulate contraction and thus the muscle remains in a relaxed state (experiments 1 and 2 in Table). From what you know about the mechanism of muscle contraction, fill in your predictions of whether the muscle will be contracted or relaxed for experiments 4, 5, and 6 (3 points, relaxed or contracted for each experiment) . Explain your answers ( 6 points, 4-5 sentences) . 6. (4 points) Why is it that intermediate filaments have identical ends and lack polarity, whereas actin filaments and microtubules have two distinct ends with a defined polarity (4 points, 2-3 sentences) ?

7. (4 points) There are no known motor proteins that move on intermediate filaments. Suggest an explanation for this observation (4 points, 2-3 sentences) . 8. (6 points) Cells expressing either N-cadherin, high levels of E-cadherin, or low levels of E- cadherin have sorted themselves out on a substratum in a cadherin-dependent manner as shown in the schematic drawing below. Which cells would you expect to correspond to white, gray, and black in the drawing, respectively (3 points, fill out table) ? Explain your reasoning (3 points, 2-3 sentences) Cells White, gray, or black? N-cadherin cells High levels of E-cadherin Low levels of E-cadherin

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9. (5 points) Cells in a developing embryo make and break gap-junction connections in specific and interesting patterns, suggesting that gap junctions play an important role in the signaling processes that occur between these cells. At the eight-cell stage, mouse embryos undergo compaction, changing from a clump of loosely associated cells to a tightly sealed ball (shown below) . You wish to know whether gap junctions are present before or after this change in adhesion. Using very fine glass micropipettes, you can measure electrical events and at the same time microinject the enzyme horseradish peroxidase (HRP), 40,000 daltons, or the fluorescent dye fluorescein, 330 daltons. Fluorescein glows bright green under ultraviolet illumination, and HRP can be detected by fixing the cells and incubating them with appropriate substrates. You obtain results at the eight-cell stage, depending on whether the injections are made immediately after cell division or later ( shown below ). Immediately after cell division, cytoplasmic bridges linger for a while before cytokinesis is completed. a. (2 points) At what stage of embryo development do gap junctions form (before or after compaction, 1 point) ? Explain your reasoning with respect to both HRP and fluorescein (1 points, 1-2 sentences) . b. (2 points) In which stage of development would you detect electrical coupling if you injected current from the HRP injection electrode and recorded voltage changes in the fluorescein electrode (before compaction or after compaction, 1 point) ? Explain your reasoning (1 point, 1 sentence).

Problem Set 3

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