Please read each question carefully. Multiple choice questions are worth four points and should be answered on the IF-AT form. A correct answer on the first try receives full credit. A correct answer on the second try receives two points. A correct answer on the third try receives one point.

Be careful to mark the correct question number on the IF-AT form.

Use the following information to answer questions 1 through 5. As part of the first Mars exploration team, you discover living single-celled organisms on the red planet! Back at home in the lab, you are surprised to see that the cells contain numerous proteins and have membranes made of phospholipid bilayers. Careful inspection of the Martian cells with a TEM shows that they have organelles which are different from any found on earth. Based on their structure, you can identify four different types of organelles which you name Groucho, Harpo, Chico, and Zeppo. You suspect that many of the organelles may be involved in the secretion of proteins from the cells.

To investigate the potential role of these organelles in secretion, you perform pulse-chase experiments similar to those of Jamieson and Palade. You first incubate some cells in a solution containing radioactive amino acids for 3 minutes. After 3 minutes, you remove a sample for TEM preparation and transfer the remaining cells to non-radioactive amino acids. The cells incubate in non-radioactive amino acids, and you remove samples at 7, 37, and 117 minutes to prepare for TEM. Autoradiography is then performed on all samples. You observe each of the four samples and count the number of silver grains found over each organelle. Your data is found in the following table.

The number of silver grains counted over each organelle.

1. What is the pathway that secreted proteins follow in the Martian cells?
1. Harpo è Zeppo è Groucho
2. Harpo è Zeppo è Groucho è Chico
3. Groucho è Harpo è Chico è Zeppo
4. Groucho è Zeppo è Harpo
2. What sort of conclusions can we make about the Chico organelle?
1. It has no function in these cells.
2. It is the last step the secretory pathway.
3. We can not make any conclusions about the Chico organelle.
4. It is not involved in the secretion of proteins and must have some other role in the cell.
3. In this experiment, when does the pulse take place? (2 pts)

During the first three minutes or when the cells are in radioactive amino acids.

4. What do the silver grains represent in the pulse-chase experiment? (4 pts)

The position of radioactive proteins in the cells. (sufficient answer)

The silver grains are formed when a radioactive emission interacts with the photographic emulsion exposing it like film. The radioactivity is given off by the radioactive amino acids that were added. The amino acids were incorporated into proteins during the pulse.

 

5. What is one isotope that you could have used to make the amino acids radioactive?_³²P, ³⁵S, ³H, ¹²⁵I, ¹⁴C (2 pts)
6. The following figure depicts three cells in the epidermis (skin).
1. Draw in intermediate filaments as they would be arranged in epidermal cells. You will need to draw a set of additional structures in these cells to accurately depict the organization of intermediate filaments. Name these additional structures. (4 pts)
2. What kind of intermediate filaments are found in epidermis? ____keratin_______ (2 pts)

 

 

3. Briefly describe the specific function of intermediate filaments in epidermal cells. (4 pts)

The high tensile strength of intermediate filaments helps to maintain the structural integrity of the epidermis by preventing overstretching of the tissue.

Since the intermediate filaments are connected to the desmosomes, they form a continuous network between the cells of the epidermis. This IF network provides elasticity and resists tearing.

 

7. SRP acquires a GTP …
1. when it floats free in the cytoplasm.
2. when it binds to the SRP-receptor.
3. when it releases from the start transfer sequence.
4. when it binds to a start transfer sequence and a ribosome.
8. SRP needs a GTP …
1. as an energy source for its movement to the RER membrane.
2. to change its conformation so that it can bind to a start transfer sequence.
3. to change its conformation so that it can bind to an SRP-receptor.
4. to change its conformation so that it can release from an SRP-receptor.
9. The cartoon below depicts three different proteins (X, Y, and Z) before they are fully processed. Some important domains within these proteins are indicated and explained in the key on the right. Based on the structure of these proteins draw them in the figure of the cell below. Draw where each protein would be located and how it would be arranged at their final destination. All membranes are shown as bilayers. (7 pts)

 

 

 

 

 

 

 

 

 

 

 

 

 

10. Vesicles from the RER fuse with the Golgi only at which compartment? ___cis Golgi___ (2 pts)
11. Where are constitutively secreted proteins sorted from proteins whose secretion is regulated? (3 pts)

At the trans Golgi network.

13. Imagine that you can purify three fractions from cells. One fraction contains only clathrin-coated vesicles; one fraction contains only COPI-coated vesicles; and one fraction contains only COPII-coated vesicles. In which fraction would you expect to find proteins that still need to have some carbohydrates added? Briefly explain your logic. (6 pts)

COPII: COPII forms vesicles at the RER for transport to the cis Golgi (2 pts). Proteins that need more glycosylation have not been through the Golgi yet, and must be on their way.

 

 

 

14. At the leading edge of a crawling cell, actin polymerization is coordinated by two different actin binding proteins to push the plasma membrane forward. A diagram of an actin filament and these actin binding proteins at the start of the process is shown below. The actin binding proteins are shown in their proper position. (Note that although an actin filament is like two strings of actin subunits wrapped around one another, I have simplified the drawing so that we are only keeping track of one strand of subunits. This is similar to the drawing I made in class.)
1. With arrows, point to the two places where actin subunits would be added. (2 pts)
2. For the two actin binding proteins, either give their names or describe their function in this process. (4 pts)

14. There are many different kinds of dynein proteins which together comprise the dynein family. The amino acid sequences that comprise the head domains of specific dyneins are very similar to one another, but the amino acid sequences that make up the tail domains are very different from one dynein to the next.
1. What are the general functions of the head domain and tail domain of a dynein? (3 pts)

Head is a motor domain which generates the force. The tail binds the cargo.

 

2. Based on your answer to A, please explain the observations about amino acid sequence stated above. (3 pts)

The heads all perform similar functions binding to microtubules, hydrolyzing ATP, and generating force, so their amino acid sequences should all be similar as well. Tails bind to different types of cargo; therefore, the amino acid sequences of the tails must vary so that they can interact with different proteins on the different cargos.

 

15. Answer either A or B. (8 pts)
1. Discuss an experiment that demonstrates that flagellar microtubule doublets slide relative to one another to produce beating.

OR

2. Briefly explain the sliding filament model of flagellar beating. Be sure to indicate which proteins generate force, what movement the force produces, and how bends are produced.

1. Isolate flagella and remove the membrane with detergent. Add ATP plus small amounts of protease. The ATP is used by dynein to generate force, and the protease cuts the nexin links. The dynein causes the doublets to slide relative to one another. Since the nexin links are cut by the protease, the doublets telescope out of the axoneme, no bending is generated.

14. The human JC virus is the cause of a fatal central nervous system disease. A recent research article in the Journal of Virology demonstrated that JC virus enters human cells via receptor-mediated endocytosis. In fact, the virus can bind to specific viral receptors that are normally found in our plasma membranes. Predict which of the following mutations would DECREASE viral entry into cells.
1. A mutant form of the viral receptor that has a lower Km than normal.
2. A mutant form of the viral receptor that has a higher Km than normal.
3. A mutant form of the dynamin protein that cannot hydrolyze GTP.
4. A mutant form of the viral receptor that lacks its cytoplasmic domain.
5. A mutant form of the viral receptor that lacks its extracellular domain.

1. i, iii, iv, v.
2. i, iii, v.
3. ii, iii, iv, v.
4. ii, iii, v.

17. Cells have both a pool of "free" or unpolymerized tubulin subunits as well as tubulin that has been polymerized into microtubules. An increase in one of these classes of tubulin, occurs at the expense of the other. Which of the following would lead to an increase in the amount of free tubulin subunits, compared to polymerized tubulin?
1. The addition of a microtubule depolymerizing drug.
2. The addition of the non-hydrolyzeable analog of GTP, GTPgS.
3. A decrease in the cellular concentration of GTP.
4. The growth of additional cilia from a cell.
5. An increase in the activity of kinesin proteins.

Use the following description and figure for questions 18-20. Nerve cells have a cell body, where the nucleus (N) is located, and a long, cytoplasmic projection called the axon. Neurotransmitters that are made in nerve cells must travel from the cell body to the axon terminal, where they are secreted from the cell via regulated secretion. One example of a neurotransmitter is acetylcholine. Nerve cells are known to have two MTOCs, which are shown below as (M), and the microtubules are represented as dotted lines.

17. If you performed indirect immunofluorescence with an anti gamma-tubulin antibody, where would you see fluorescence?
1. Along the length of the microtubules.
2. Along the length of the microtubules AND at the two MTOCs.
3. At the two MTOCs.
4. At the axon terminal.
18. What motor protein would be used to move acetylcholine from the cell body to the axon terminal?
1. Dynein
2. Kinesin
3. Myosin.
4. Keratin.
19. If you had a mutant strain of nerve cells that lacked T-SNARES on their plasma membrane, what would happen to acetylcholine?
1. Acetylcholine would be secreted the same as in normal cells.
2. Acetylcholine would be incorrectly targeted to the lysosome.
3. Acetylcholine would accumulate in COPII coated vesicles in the cell body.
4. Acetylcholine would accumulate in vesicles at the axon terminal.

 

20. Answer either A or B. (8 pts)
1. Briefly describe the role that GTP and GDP play in the control of microtubule dynamic instability.

OR

2. Briefly outline part of the crossbridge cycle of myosin starting at the hydrolysis of ATP and with myosin that has ADP bound.

1. In order to polymerize, the plus end of a microtubule must have tubulin subunits with GTP bound. This is the GTP cap.

After polymerization into a microtubule, the tubulin will hydrolyze GTP into GDP.

GDP-tubulin wants to disassemble from a microtubule.

If the plus end of a microtubule has only GDP-tubulin bound, the microtubule will swithch to a rapid depolymerization phase.

2. Hydrolysis of ATP into ADP and Pi causes a conformation change in the myosin head so that the head swings toward the plus end of actin, "cocking" the head. In addition the myosin now weakly binds to actin.