BIOLOGY 285 Study Guide - Quiz Guide: Tubulin, Kinesin, Microscope Slide
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Bio285 F19 Problem Set 3 Francis
Dynamic instability causes microtubules either to grow or to shrink rapidly. Consider an
individual microtubule that is in its shrinking phase.
A. What must happen at the end of the microtubule in order for it to stop shrinking and start
A microtubule in its shrinking phase has lost its GTP cap and the tubulins at the ends are
in the GDP form. In order to stop it from shrinking and start growing, enough GTP-subunits
must be added quickly to regain its GTP cap. That is, the rate of tubulin addition needs to
exceed the rate of GTP-hydrolysis (replaces GTP with GDP).
B. How would an increase in the tubulin concentration affect this switch from shrinking to
The rate of GTP-tubulin addition will be greater at a higher concentration of tubulin.
There are more free tubulins available to add to the growing microtubule and it will start to
shrink again when GTP hydrolysis catch up to GTP-tubulin addition.
C. What would happen if GDP, but not GTP, were present in the solution?
If GDP was the only thing present in the solution, then the microtubule will continue to
shrink and disappear. This is because tubulin dimers with GDP have very low affinity with each
other and will not be stable when added to microtubules.
D. What would happen if the solution contained an analog of GTP that could not be
If the solution contained a non-hydrolysable GTP, microtubules would continue to grow
until all free tubulin subunits have been used up.
Question 2: Microtubule Polymerization Assay
Please answer the following questions about this graph of a microtubule polymerization assay:
A. How is this experiment performed (i.e. what is added, what is measured, etc?)
Bio285 F19 Problem Set 3 Francis
This is an experiment on kinetics of microtubule assembly in vitro performed with
purified tubulin and GTP. It looks at the assembly rate and dynamics overtime of pure tubulin.
The tubulin and GTP are added to the tube and a spectrophotometer are used to measure the
concentration of microtubule growth. A higher absorbance reading means more light is being
absorbed, which means more microtubules. However, this only gives you the total amount of
polymerization since you can’t use this method to determine the actual length of the
B. What is the period “A” called? Briefly describe what molecular events are happening during
Period A is called the lag phase. During this time, tubulin dimers aggregate into
oligomers which serve as nuclei for further microtubule growth. Here, the tubulin dimers need
time to bind and form oligomers and protofilaments. Then, they form a full sheet and the
closing of the sheet forms the cylindrical microtubule.
C. What is the period “B” called? Briefly describe what molecular events are happening during
Period B is called the elongation phase. This is when tubulin dimers are added to the
exposed ends of the growing filament, causing filament elongation. The addition of tubulin
occurs at both ends, but it grows much faster at the plus end of the microtubule.
D. What is the period “C” called? Briefly describe what molecular events are happening during
C is called the plateau or equilibrium phase. The microtubule reaches a point where the
amount of free tubulin is diminished, and the assembly is balanced by disassembly. Here, the
rate of tubulin addition and depolymerization reaches equilibrium.
Question 3: Gliding assay
To make microtubules for a gliding assay, you add fluorescent GTP-tubulin to your test tube, as
well as taxol. Then you leave the microtubules sitting on your bench-top for the night and the
next day you perform your gliding assays.
A. Why is it important to use taxol when you make microtubules for your gliding assay? In your
answer, make sure you include an explanation of the gliding assay – how it is done, and what
it’s used for.
In gliding assays, double-sided tape is used to create a small channel between a
microscope slide and cover slip. A flow purified kinesin is placed into the channel, where they
will stick to the microscope slide (the kinesin “feet” will stick up into the channel so
microtubules can stick to them). From this experiment, you are able to see what direction
motor proteins walk in and how fast motor proteins walk along microtubules. It is important to
use taxol because it prefers to bind to polymerized microtubules and it turn, will stabilize the
structure. We want the microtubule to stay still in gliding assays so the motor protein can walk