A5

Question 1

You are interested in studying kinesin movements. You therefore prepare silica beads and coat them with kinesin molecules so that each bead, on average, contains only one kinesin molecule attached to it. You add these kinesin-coated beads to a preparation of microtubules you have polymerized. Using video microscopy, you watch the kinesin move down the microtubules.

Kinesin-GFP has been measured to move along microtubules at a rate of 0.3 µm/second, and single-molecule studies have revealed that kinesin moves along microtubules progressively, with each step being 8 nm. How many steps can the kinesin molecule take in 4 seconds, assuming that the kinesin stays attached to the microtubule the entire 4 seconds? [Blank1]

Answer 1

150

0.3um = 300nm
in 4 seconds = 1200nm
step size = 8nm therefore
1200nm/8nm per step = 150

Question 2

Since each kinesin molecule is thought to take approximately 100 steps before falling off the microtubule, will you see your silica beads detach from the microtubule during your 4 seconds of observation?

Answer 2

yes

(because each kinesin will take more than 100 steps in the time frame)

Question 3

What would you predict would happen to the kinesin-coated silica beads if you were to add AMP-PNP (a nonhydrolyzable ATP analogue)?

Answer 3

the beads should stop moving - no longer see them moving down the microtubule - one molecule of ATP is hydrolyzed per step that kinesin takes - without ATP hydrolysis, translocation of the beads will be inhibited
you may still see the beads associated with the microtubules because AMP-PNP does not inhibit the association of kinesin with the molecule

Question 4

Before chromosome segregation in M phase, the chromosomes and the segregation machinery must be appropriately prepared. Indicate whether the following statements are true:

Microtubule-dependent motor proteins and microtubule polymerization and depolymerization are responsible for the organized movements of chromosomes during mitosis.

Each microtubule-organizing center contains a pair of centrioles and hundreds of γ-tubulin rings that nucleate the growth of microtubules.

Answer 4

both true

Question 5

What is the function of the following?
- aster or astral microtubules
- interpolar microtubules are stabilized by interactions with ______ via _____
- kinetochore microtubules ________to contribute to _________

Answer 5

2. Aster or astral microtubules interact with the cell cortex
3. Interpolar microtubules are stabilised by interactions with each other via motor proteins
4. Kinetochore microtubules depolymerize to contribute to chromosome movement in anaphase A

Question 6

Disassembly of the nuclear envelope in animal cells______________

Answer 6

must occur in order for kinetochore microtubules to form in animal cells

Question 7

an antibody against motor proteins that move from the minus end of microtubules towards the plus end (i.e. plus-end directed motors) would impair anaphase __ but not anaphase ___

Answer 7

B
A

Question 8

Last summer, while working in the laboratory of Professor Mike Rotubulis, you discovered a new protein that regulates microtubule dynamics. First, you isolated proteins from a cellular extract that bound to a tubulin affinity column. You then separated the proteins from each other by loading the mixture of proteins on an ion-exchange column, eluting the column with increasing salt concentration and collecting small “fractions” of protein as they dripped from the column. To test if each fraction contained microtubule regulators, you mixed it with fluorescent tubulin and purified centrosomes and analyzed the reaction microscopically to measure the size of the aster formed. You found that fractions 8, 9, and 10 promoted formation of an unusually large aster, consisting of long microtubules. Because electrophoretic separation of the fractions on an SDS-PAGE gel revealed a plentiful protein with an apparent molecular weight of 98 kD, you named the protein p98.

Does p98 behave like a MAP or a microtubule motor? Hint: does p98 promote or inhibit microtubule growth?

Answer 8

MAP
as it promotes microtubule growth

Question 9

these events occur in what stage?
exocytosis and endocytosis stop
chromosomes attach to and move along spindle microtubules
the golgi apparatus fragments

Answer 9

pro-metaphase

Question 10

Which of the following statements is false

The cleavage furrow…

1.	 always forms parallel to the interpolar microtubules

2.	 is a puckering of the plasma membrane caused by constriction of a ring of filaments attached to the membrane

3.	 forms during telophase

4.	 will not begin to form in the absence of a mitotic spindle

Answer 10

1 (is perpendicular)