Cell Structure and Function                                                       Name___________________

Homework 6                                                                Collaborators___________________

Due: Wednesday, December 5, 2001

 

 

            A company involved in raising fish has hired you commercially.  The company has recently lost large amounts of fish from its pond due to a large community of alligators, which consider fish their main dish.  You cannot kill the alligators because environmental laws protect them.  Your supervisor has heard about fish that are able to change their color, and thereby escape predators, by moving pigment granules in their epithelial cells.  He suggest that genetic engineering techniques could be employed which would give the company’s fish this protective coloration and make them invisible to the alligators.  Your supervisor suggests you find more about these pigment molecules.  

 

            In order to learn more about this process, you purchase several fish that are capable of changing colors.  You isolate a few epithelial cells and view them under a microscope.   You notice that the pigment granules are dispersed throughout the cell.  After adding epinephrine (the “fight or flight” hormone) to the cells, the pigment granules begin to aggregate.

           

 

 

 

 

 

 

 

 

 

 

Your boss asks what the pigment molecules are moving on?  You tell your boss that this type of pigment granule movement occurs on microtubules.  To show off your knowledge of microtubules you draw a diagram to indicate the expected polarity of the microtubules in these cells:

 

 

 

 

 

 

 

 

 


1.  Show the plus (+) and minus (-) ends of the two microtubules in this diagram. (2 points)

            Your supervisor suggests that the movement of pigment granules is the result of the dynamic instability of MT’s (i.e. aggregation is due to MT shrinkage and dispersal is due to MT elongation).  You argue that movement is more likely due to motor proteins and suggest a simple experiment to discriminate between your idea and your supervisor’s hypothesis.  You label an unstimulated cell with a fluorescent anti-tubulin antibody and observe the following distribution of MT’s (for simplicity only three MT’s are shown).

 

            You then treat another batch of cells with epinephrine to trigger aggregation.  Once the pigment granules are tightly aggregated you label the cell with the same fluorescent antibody.  In the cells below show the expected pattern of the three MT’s if your supervisor’s hypothesis is correct compared to the pattern expected if your hypothesis is correct.  (6 points)

 

            As expected, the fluorescent labeling experiment supports your ideas.  Therefore, you decide to examine the roles of motor molecules in this system.  You suggest to your boss that two separate families of MT motors may drive the movement of the pigment granules.  Based on your knowledge of the motors involved in other types of MT-based motility: (4 points)

 


3.  What motor is most likely involved in:

 

            Dispersal: ______________________

 

            Aggregation:____________________

 

            To test your idea, you purify the proteins from a batch of pigment cells.  After separating the proteins and testing them in the MT sliding assay designed by Vale and colleagues (motor proteins absorbed to glass bind to and move single MT’s in the presence of ATP; movement is visible by computer-enhanced light microscopy) you identify two prospective motor proteins.  To determine the directionality of the motors, you use demembranated flagellar axonemes in place of MT’s in the gliding assay.  These axonemes retain the 9+2 arrangement of MT’s and splay apart at the tip as show below.  This allows you to determine the direction of sliding relative to the polarity of the microtubules in the axonemes.

 

  1.  In the diagram below, use an arrow to show the direction the motor involved in the pigment granule aggregation in the fish cell would cause the axonemes to move in the gliding assay.  (4 points)

 

            Your supervisor is mightily impressed with your skill and asks if you recall anything from your undergraduate days about how epinephrine might turn motor proteins on and off.  You remember a lecture or two on signal transduction in which you learned that epinephrine turns on a G-protein, which turns on adenylate cyclase, then cAMP-dependent protein kinase and then glycogen phosphorylase.  Your notes from these lectures include two vague comments: first, that cAMP-dependent protein kinase also has other targets and second, that phosphorylation usually, but not always, activates the target protein.  You do two experiments.  In the first, you find that epinephrine –treatment of the cells causes an increase in cytosolic cAMP concentration (i.e. the epinephrine is signaling via the usual route).  In the second, you find that epinephrine-treatment causes phosphorylation of kinesin, but not cytoplasmic dynein.  You also find that when the epinephrine is removed the kinesin is dephosphorylated. 

 

            Based on these data, develop a plausible hypothesis about how epinephrine-mediated phosphorylation may control motor-based pigment granule movement in these cells. (4 points)