Q1. Here are three scenarios to consider in the pump metaphor: a broken pump, a

Question

Q1.

Here are three scenarios to consider in the pump metaphor: a broken pump, a blocked turbine, and a leaky hose. The questions below ask you to determine how would you expect the water pressure in the hose to change in each scenario. Your answers will help you understand how membrane potential is regulated.

Here is the diagram of the water hose metaphor again to use as you answer the questions.

Scenario 1: Broken Pump

1 point possible (graded)

Scenario 1: What would happen to the hose pressure if the pump broke down?

Select ONE option:

1. The pressure will not change because both the rate of water pumping into the hose and the rate of water flowing out of the hose will decrease.

2. The pressure will decrease because the rate of water pumping into the hose will decrease before the rate of water flowing out of the hose significantly decreases.

3. The pressure will increase because the rate of water pumping into the hose will increase while the rate of water flowing out of the hose will remain the same.

Scenario 2: What would happen to the hose pressure if you blocked the turbine?

Select ONE option:

1. The pressure will increase because the rate of water pumping into the hose will remain the same while the rate of water flowing out of the hose will decrease

2. The pressure will not change because both the rate of water pumping into the hose and rate of water flowing out of the hose will decrease.

3. The pressure will decrease because the rate of water pumping into the hose will decrease while the rate of water flowing out of the hose will remain the same.

Scenario 3: What would happen to the hose pressure if you cut holes in the hose?

Select ONE option:

1. The pressure will increase because the rate of water pumping into the hose will stay the same while rate of water flowing out of the hose will decrease.

2. The pressure will not change because both the rate of water pumping into the hose and rate of water flowing out of the hose will decrease.

3. The pressure will decrease because the rate of water pumping into the hose will stay the same while the rate of water exiting the hose will increase.

Q2.

Recall that tetramethylrhodamine (TMR) is a fluorescent, positively-charged small molecule. It is used to measure mitochondrial electrical potential, by measuring the extent to which the TMR molecule enters mitochondria. The brightness of the signal from TMR is directly related to the negative charge in the matrix.

Given your exploration of how these compounds affect membrane potential, how would you expect them to affect the TMR signal in mitochondria?

Part 1. The addition of oligomycin (an ATP synthase inhibitor) will ----------- the TMR signal.

Select ONE option for the blank:

1. increase

2. decrease

Part 2. The addition of FCCP (a protonophore that allows leakage of protons across the membrane) will ---------------- the TMR signal.

Select ONE option for the blank:

1. increase

2. decrease

Part 3. The addition of cyanide (a complex IV inhibitor) will ---------------- the TMR signal.

Select ONE option for the blank:

1. increase

2. decrease

Q3.

Your lab has recently identified a small molecule present in e-cigarette vapor called TX32. You suspect it may be toxic and want to test its effects on mitochondrial function and membrane potential more specifically.

You prepare four dishes with cells. You treat the cells with three different doses of TX32 and leave one untreated for comparison (Figure A).

The following day, you add a small amount of TMR for 1 hour (Figure B) and use a fluorescence microscope to measure the TMR signal in each dish (Figure C).

Figure A. Dishes of treated and untreated cells.

Figure B. Addition of TMR to each cell culture dish.

Figure C. Example of TMR signal in a cell.

Membrane Potential Experiment

1 point possible (graded)

You observe a significant decrease in TMR signal in TX32-treated cells that varies by dose (Figure A). How might TX32 affect membrane potential?

Figure A. TMR signal in treated and untreated cells.

Select ONE option:

1. Directly inhibits ETC proton pumping activity like rotenone or antimycin.

2. Interfere with glucose metabolism and/or TCA cycle and thereby reduce NADH and FADH2 supply to ETC

3. Increase the proton leak through the membrane like FCCP.

4. All of the above.

Explanation / Answer

Please find the answers below:

Answer 1:

Part 1: Choice 2 (as the outward flow of water will decrease, the inward flow of water too shall decrease leading to significant loss in water pressure in the hose)

Part 2: Choice 1 (as the outflow is decreased but inflow is maintained, the large volume of water in the hose will increase its pressure)

Part 3: Choice 3 (there would be overall decrease in hose pressure since the outflow volume is higher than inflow volume)

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