cell biology exam #3 - carpet cleaning mobile, al · zoo 3113-002 fall 2009 exam #3 student id#...

Cell Biology Exam #3 Fall 2009

ZOO/BOT/MBIO 3113-002

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You must answer 4 of the first 5 questions.

Once you complete your answers to the 4 required questions, you may answer the Extra Credit question

We will grade only 4 questions from among the first 5. If you begin to answer a question and then decide to skip to another, you must indicate

which question is NOT to be graded by writing boldly DO NOT GRADE on the exam page you do not wish to have graded.

Note: Each question is worth 20 points. There are 80 points on the exam (plus 6 Extra credit points).

ZOO 3113-002 Fall 2009 Exam #3 Student ID#

Question #1.

You have isolated a bacterial transporter that transports Mg 2 and Ca2 in opposite directions. You produce lipid vesicles that contain this Mg 2 �C2 pump as the sole membrane protein. The Mg 2 �C2 pumps are oriented such that Mg2 is pumped into the vesicle while C2 is pumped out. Each pump transports one Mg 2 ion into the vesicle and one C2 ion out of the vesicle during each pumping cycle and ATP hydrolysis powers this transport, as illustrated in the figure below.

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A. Predict what would happen if the solution inside the vesicles contained 75 mM Mg2 and 75 mM Ca2 ions, while the solution outside the vesicles contained 75 mM Mg2 and 75 mM C2 ions as well as 1 mM ATP, and you had a 10,000 fold excess of fluid outside the vesicles compared with the volume inside the vesicles. Specifically, be sure to state whether you think there would be net movement of ions and if so, how the concentrations of the Mg 2 and Ca24 ions inside and outside the bilayer would change. In addition, be sure to state whether you would be creating an electrical gradient, a chemical gradient, or both. (Note: the typical

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2+ 2 intracellular [Ca ] s 10 -7 M; typical extracellular [Ca ] is about 1mM. Thus, you are unlikely to deplete Ca + from either vesicles or the fluid. 1mM ATP is also an excess of ATP). (10 points)

B. Explain how long the pump will run under these conditions. (5 points)

C. How would the sequence of ions binding on a symporter differ from that of ions binding to the antiporter described above? (5 points)

ZOO 3113-002 Fall 2009 Exam #3 Student D# I I Question #2. You are interested in understanding how the neuromuscular junction works. The normal voltage-gated Na channel has the characteristics depicted by the solid line in Figure Q2A. You decide to replace this channel with a mutant voltage-gated Na’ channel with the characteristics depicted by the doffed line. Do you predict that the cell containing the mutant Na channel will require more, less, or the same amount or acetylcholine to be released from the nerve terminal to stimulate muscle contraction? Explain your answer. (10 points)

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A neuron can receive inputs from many different presynaptic cells and needs to integrate information about the excitatory postsynaptic potentials (PSPs) and inhibitory PSPs it is receiving. Different types of K 4 channels are required to ensure that the frequency of action potentials reflects the magnitude of the combined PS Ps. If a normal cell produces a pattern of membrane depolarization following the combined PSPs illustrated in Figure Q2B, how would a drug that reduces the activity of the delayed K channels alter the pattern of membrane depolarization? Explain. (10 points)

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ZOO 3113-002 Fall 2009 Exam #3

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Question #3. The olfactory receptor neurons in frogs resemble those of mammals, in that they express olfactory receptors that are coupled to a trimeric G protein. When the G protein is activated, it activates an adenylyl cyclase to produce cAMP, which then opens cyclic-AMP gated cation channels in the plasma membrane. The opening of these channels depolarizes the membrane, leading to the production of an action potential. Your friend is interested in why neurons stop responding to an odor after prolonged exposure to it, a process called adaptation. He has conducted experiments examining the depolarization of the olfactory receptor neuron, the binding of odorant to the receptor, the activation of the G protein, the levels of cAMP in the cell, and the phosphotylation of adenylyl cyclase. His results are summarized ii1 the able below.

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From these results, what step in the odor transduction pathway is altered to elicit adaptation? Propose a possible molecular mechanism to explain why olfactory neurons no longer respond to an odor after prolonged exposure to it. (10 points) i-k

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Question #4. You are studying a protein that normally resides in the plasma membrane. The organization of this protein is depicted in Figure Q4, where the gray boxes labeled A, B, and C represent transmembrane regions and the segments labeled 1, 2, 3, and 4 represent the portions of the protein between the transmembrane regions.

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If you also know that segments 2 and 4 contain N-linked oligosaccharides, draw the topology of the protein as it resides in the plasma membrane (how is the protein oriented in the membrane?). Be sure to label the cytoplasmic and extracellular face of your membrane and the N-terminus and C-terminus of your protein. (10 points) c pLc cQ

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Do positively charged amino acids flanking the initial start-transfer signal on the polypeptide sequence at transmembrane region A precede it, or follow it within the polypeptide s e? Explain your reasoning. (10

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ZOO 3113-002 Fall 2009 Exam #3 Student ID# I /1 1 l Question #5. Protein kinase A (PKA) is a serine/threonine kinase that phosphorylates the nuclear CREB protein to initiate target gene transcription. Increased PKA activity is always coupled to the activation of a transmembrane G-protein coupled receptor (GPCR). The activity of another serine/threonine kinase, protein kinase C (PKC) can also be activated by GPCRs or alternatively by a receptor tyrosine kinase (RTK). Explain how PKA activity is restricted to GPCR activation, but PKC activity can be regulated by either receptor type, GCPR or RTK. (10 points)

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Extra credit (6 points)

Phosphoinositides are minor membrane phospholipids that are increasingly understood to play major roles in many cell functions. Modifications to phosphatidylinositol (P1) by different PT kinases give rise to phosphoinositides that vary widely in their functions. List and explain 6 membrane-associated cell processes that depend on changes to, or interactions with, specific membrane phosphoinositides. For each example, be sure to indicate the phosphoinositide involved, how activity on the phosphoinositide is mediated (e.g., are particular enzymes involved?), and what is the "downstream" effect within the cell resulting from the change to or interaction with the specific phosphoinositide. (1 point each, up to 6 points total)

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Note: There are only 75 points on the exam (plus 5 points for the extra-credit question). You riuist answer Questions I and 2; you may chose to answer 3 (and only 3) questions from among the 4 questions worth 10 points each (Questions 3-6); the extra credit question (worth 5 additional points) is optional.

Exam #4 Zoology 3113-002

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Question #1(25 points) You must answer this question You have discovered a new cytoskeletal polymer that resembles actin in an archaean; you call the subunit for this polymer AALP (for archaea actin-like protein). AALP binds to ATP and can catalyze the hydrolysis of ATP to ADP. As with actin, this hydrolysis occurs more quickly when AALP is in a filament and most of the free AALP is bound to ATP. You measure the rate of AALP filament growth in vitro at the plus (more quickly growing) end and at the minus (more slowly growing) end and obtain the graph diagrammed in the figure below.

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CapZ, would the presence of CapZ protein change the growth characteristics at either the plus or minus end, or both? Why? (5 points)

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/ F. As with actin, AALP subunits may exist in two forms, the T form and the D form. Where would you expect to find more of the T form of the AALP subunits on newly polymerized filaments? (5 points)

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Exam #4 Zoology 3113-002 Student IM

Question #2 #2 (20 points total) You must answer this question You have isolated a strain of mutant fission yeast that divides normally at 30°C but arrests in the cell cycle before M phase at 37°C. These mutant yeast cells are not defective in the production of MCdk, because you have isolated the mitotic cyclin and mitotic Cdk7im these mutant yeast and find thatboth Droteinsare

complex at both temperatures. Which among the following types of mutations could be responsible for the behavior of this strain of yeast? Explain why each of the possibilities listed below either can or cannot explain the observed cell cycle arrest at the higher temperature. (Note: More than one of the possibilities listed below may provide a plausible explanation for the observed cell cycle arrest). (5 points each)

A. Inactivation of an enzyme that ubiquitylates M�cyclin. B. Inactivation of the Wee kinase. C. Inactivation of the CAK kinase. D. The continuous production of a phosphatase that removes all phosphate groups from the M�Cdk.

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Question #3 (10 points) You may choose to answer this question You have obtained an antibody to myosin that prevents the movement of myosin molecules along actin filaments. If this antibody were injected Into cells, would you expect the movement of chromosomes at anaphase to he affected? Flow would you expect antibody injection to affect cytokinesis? Explain your answers.

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Question #4 (10 points) You may choose to answer this question

In yeast, pheromone-induced activation of the mating factor receptor leads to activation of the monomeric

GTPase Cdc42, followed by rearrangements in the actin cytoskeleton. Some changes are brought about by

the activation of another monomeric GTPase Rho I. targeting increased activity of formins. It is now also

known that ARP complex proteins are indirect targets of Cdc42 activity and both ARP complex proteins

and formins promote actin polymerization. Explain how the activation of these two different types of actin

polymerization-enhancing molecules (ARPs and formins) by the same receptor could lead to different rearrangements of the actin cytoskeleton.

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Exam #4 Zoology 3113-002

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Question #5 (10 points) You may choose to answer this question Your friend comes to you in a panic. He was purifying extracts from interphase cells as well as mitotic

cells. Unfortunately, the labels came off his tubes and he cannot tell which extract is from which cells. You

do an experiment in which you add a small amount of each extract to fluorescent microtubules you have polymerized in vitro, and then use video microscopy to follow the behavior of individual microtubules in the reaction over time. Your results are shown in the graphs below.

microtubules polymerized in the presence of Extract #1

microtubules polymerized in the presence of Extract #2

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Which extract do you think is from mitotic cells and which from interphase cells? (5 points) Why? (5 points)

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Exam #4 Zoology 3113-002 Student ID# \ ’Z Y

Question #6 (10 points) You may choose to answer this question Neurons contain two general types of projections. Incoming signals are received on dendritic projections, whereas signals transmitted to other cells are sent to the nerve terminal along an axonal projection. Vesicles containing neurotransmitters and receptors are transported along microtubules contained in these projections, using the motor proteins dynein and kinesin. You perform an experiment in which you express a GFP-labeled kinesin that glows green under a UV light. You note that within axons, cargo vesicles attached to the fluorescently labeled kinesin travel only from the nerve cell body toward the nerve terminal. Interestingly, you note that within dendrites, cargo vesicles attached to the labeled kinesins are moving bi-directionally, both toward the terminal ends of the dendrites, and back toward the cell body. How can you explain this result?

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Exam #4 Zoology 3113-002 Student ID#

Extra Credit (5 points total) You have identified a drug, 1D555, that causes M�Cdk protein levels to remain high in the cell. You have two favorite models for how 1D555 works. Model 1: 1D555 activates a transcription factor that stimulates the genes encoding M�cyclin and M�Cdk, so that excess M�Cdk protein is produced. Model 2: 1D555 inhibits the activity of the Cdc20�APC/C complex that normally targets M�Cdk for destruction.

Your friend works in a lab with many fancy microscopes and offers to examine cells that are treated with drug. She sees that cells treated with 1D555 assemble a mitotic spindle and their chromosomes seem to align at the metaphase plate. However, the chromosome segregation seen during normal anaphase does not occur. Does this new information cause you to favor model 1 or model 2? (2 points) Explain. (3 points)

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You must answer questions 1-4.

You may answer Question #5 for a maximum of 5 extra credit points

Exam 3. Zoology 3113-002 Student ID#

Question #1 (5 points each; 25 points total)

You are studying a protein that has two transmembrane domains. The spacing of the transmembrane domains is shown as gray boxes in the Figure. The C-terminal domain of this protein is cytoplasmic.

A. Is the N-terminal domain of the protein cytoplasmic or extracellular? Why?

B. Where do you expect to find the N-terminal domain located as the N

protein is shuttled in a transport vesicle to the plasma membrane?

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C. Your friend discovers that this protein is glycosylated. What information did she use to determine the protein is likely to be

C glycosylated (all she had available was a text file of the amino acid J - sequence you sent her for study)? Which region of this protein is most likely to be glycosylated? Explain.

D. How could you modify the protein so that the orientation of the C-terminal domain is extracellular, rather than cytoplasmic?

E. Would this change to the protein affect your answer in part C? If so, how?

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Question #2. (30 points total; 6 points each section) Cholesterol is an essential component of the plasma membrane, but people who have very high levels of cholesterol in their blood (hypercholesterolemia) tend to have heart attacks. Blood cholesterol is carried in the form of cholesterol esters in low-density lipoprotein (LDL) particles. LDL binds to a high affinity receptor on the cell surface, enters the cell via receptor-mediated endocytosis, and ends up in lysosomes. There its protein coat is degraded, and cholesterol esters are released and hydrolyzed to cholesterol. The released cholesterol enters the cytosol and inhibits the enzyme HMG CoA reductase, which controls the first unique step in cholesterol biosynthesis. Patients with severe hypercholesterolemia cannot remove LDL from the blood. As a result, their cells do not turn off normal cholesterol synthesis, which makes the problem worse. LDL metabolism can be conveniently divided into three stages experimentally: binding of LDL to the cell surface, internalization of LDL, and regulation of cholesterol synthesis by LDL. Skin cells from a normal person and a patient suffering from severe familial hypercholesterolemia (DM) were grown in culture and tested for LDL binding, LDL internalization, and LDL regulation of cholesterol synthesis. The results are shown in Figure 2 below.

A. In Figure 2A, the surface binding of LDL by normal cells is compared with LDL binding by cells from patient DM. Binding appears to be unaffected in the cells from patient DM. Why does binding by normal cells and by DM’s cells reach a plateau?

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Exam 3. Zoology 3113-002 Student ID# - D2fl

Question #3. You are interested in cell shape and discover that signaling through a GPCR is important in controlling cell shape in cultured rat fibroblasts. The G protein downstream of this receptor activates adenylyl cyclase, which ultimately leads to the activation of PKA. You find that the membrane of cells that lack the GPCR are 15% less ruffled than normal cells, whereas cells that express a mutant, constitutively activated version of PKA are 15% more ruffled than normal cells. Furthermore, the normal fibroblasts become less ruffled when treated with cholera toxin, which has been shown to inhibit certain subclasses of the a subunit of the G protein. Given these results, what do you predict would happen to the cell’s shape (more ruffled, less ruffled, or no change) if normal cells were treated in the following fashion? Explain the reasoning behind each of your answers. (5 points each; 25 points total)

A. You add GTPyS (a non-hydrolyzable form of GTP). B. You inhibit phospholipase C with the PLC inhibitor neomycin. C. You add staurosporine, a drug that specifically inhibits the activity of protein kinase C. D. You add a drug that inhibits adenylyl cyclase. E. You add dibutyrl-cAMP, a membrane-permeable analog of cAMP.

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Question #2 (continued) C. In Figure 2C, the regulation of cholesterol synthesis by LDL in normal cells is compared with that in cells from DM, and LR cells expressing the DM LDL receptor. Why does increasing the external LDL concentration inhibit cholesterol synthesis in normal cells and LR cells, but only slightly in cells from DM?

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D. How would you expect the rate of cholesterol synthesis to be affected if normal cells and cells from DM were incubated with cholesterol itself? (Free cholesterol crosses the plasma membrane by diffusion.)

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Question #4. Phosphoinositides are minor membrane phospholipids that are increasingly understood to play major roles in many cell functions. Modifications to phosphatidylinositol (P1) by different P1 kinases give rise to phosphoinositides that vary widely in their functions. List and explain five (5) membrane-associated cell processes that depend on changes to, or interactions with, specific membrane phosphoinositides. For each example, be sure to indicate the phosphoinositide involved, how activity on the phosphoinositide is mediated (e.g., are particular enzymes involved?), and what is the "downstream" effect within the cell resulting from the change to or interaction with the specific phosphoinositide. (5 points each; 25 points total)

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Extra credit Question #5. (5 points) MAP kinase signaling regulates multiple cell functions. For example, the yeast mating response is determined by activation of a receptor coupled to MAP kinase signaling. The same yeast cell also activates MAP kinase signaling in response to osmotic changes to its environment. Both of these responses depend upon activation of the same MAP kinase kinase kinase (MAPKKK) protein but different downstream processes are subsequently activated (i.e., the mating response or glycerol synthesis). How are these differential responses mediated following activation of the same MAPKKK within the same cell type?

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cell biology exam #3 - carpet cleaning mobile, al · zoo 3113-002 fall 2009 exam #3 student id#...

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