BIS104
Winter 2010
Problem Set 4
“Due” March 1.
This problem set will not be collected, but you should do it before looking at answers.
Answers will be posted on March 1.
1-4. You have microinjected a newt lung cell with rhodamine-labeled tubulin, cagedfluorescein labeled tubulin and the fluorescent DNA stain, DAPI. You wait until the cell
enters metaphase and you uncage a bar of caged-fluorescein tubulin in the spindle. The
figures below show the fluorecence intensity of uncaged fluorescein on the y axis and
the distance from the left spindle pole (centrosome) on the x axis. The position of one
sister chromatid that is attached to the left pole by a kinetochore fiber is also shown.
1. What is the rate of poleward flux in m min-1? 0.5 μm/min
2. How will the rate of poleward flux be affected by the addition of taxol?
Taxol will stabilze GDP-tubulin subunits and inhibit depolymerization. Since
poleward flux requires loss of tubulin subunits at the minus end of the microtubule,
poleward flux will be inhibited by taxol.
3. What will happen to the distance between the centrosome and the chromosome if
you wait for 1 hour in the presence of taxol?
A. Grow
B. Shrink
C. No change
C. Taxol inhibits chromosome segregation and therefore there will be no
net movement.
4. If you microinjected the cell with cyclinB 90 protein during metaphase, what would
happen to the distance between the centrosome and the chromosome after waiting for
1 hour?
A. Grow
B. Shrink
C. No change
C. Deletion of destruction box leads to the stabilization of cyclin B and the arrest
of cells in mitosis. Since the forces holding chromosomes at the metaphase plate
are in balance, there will be no net movement of chromosomes in the presence of a
cyclin B 90.
5. If you microinjected the cell with a polypeptide consisting of the first 90 amino acids
of cyclinB during metaphase, what would happen to the distance between the
centrosome and the chromosome after waiting for 1 hour?
A. Grow
B. Shrink
C. No change
B. This peptide will be degraded and cannot bind to CDK to influence its activity,
therefore after 1 hour endogenous cyclin B will be destroyed, anaphase will occur
and chromosomes will move closer to the centrosome.
VIDEO ASSIGNMENT
Watch PART 2 Dick McIntosh’s iBioseminar on “Unserstanding Mitosis Through
Experimentation.” Answer the following questions. The video is about 40 minutes. It
focuses on experiments on mitosis. Go to the following website:
http://www.ibioseminars.org/index.php?option=com_content&view=article&id=70&Itemid=70
6. Describe kinesin-5. What is its domain structure? How can cell biologists inhibit it?
What happens when you inhibit kinesin-5?
Kinesin-5 plus-end directed MT motor and forms as a homotetramer. It crosslinks anti-parallel MT in the middle of the spindle. Genetic or pharmacological
inhibitors (eg monastrol) inhibit spindle formation. Mono-polar spindles form.
Kinesin-5 also provides the forces that push the opposite poles apart.
7. Are chromosomes required for bi-polar spindle formation? How was this found?
No! If you physically remove the chromosomes with a micro needle from a spindle,
the spindle stays in tact and division still occurs!
8. What other kinesins are involved in spindle formation? And what is special about
kinesin-14? What is special about Kinesin 13?
Kinesin-14 is a minus end MT motor (only minus-end directed). Kinesin-14 balances
out the forces of Kinein-5 to maintain the length of the spindle.
Kinesin-13 is a depolymerizer. It doesn’t have motor activity. One kinesin-13 acts
at the poles removing subunits at the minus ends. Then plus ends grow, creating
flux. Meanwhile kinesin-5 pushes MT towards the poles.
9. How was treadmilling or flux observed in original experiments?
Fluoresence speckle microscopy.
10. What motor is at the cortex that pulls the spindle poles apart in some cells?
Dynein, which is minus-end directed. So kinesin-5 in the middle and dynein at the
cortex are doing the same thing.
11. How is dynein targeted to the kinetochore?
Dynein is recruited to the kinetochore by the ROD/ZW10 complex. Remember that
motors need to be targeted to specific cargos. Here the cargo is the kinetochore
and chromosome, and dynein is targeted there by ROD and ZW10. Interestingly,
in a zw10 mutant fly, most chromosomes segregate properly, even without Dynein.
So when Dr. McIntosh says its hard to know the role of dynein at the kinetochore
for chromosomal segregation and tension, I would argue that the zw10 mutant
studies suggests dynein is NOT the answer. If you are interested in more details
on how dynein is targeted to the kinetochore, see Prof. Starr, as his PhD thesis
focused on the role of ZW10 and targeting of Dynein to the kinetochore.
12. What is the polar elimination force?
The polar elimination force pushes chromosomes away from the poles to help
congression to the metaphase plate during prometaphase. The prime candidate for
this is a chromokinesin, which is attached to chromosomes and move toward the
plus-ends (the center of the spindle).