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Tutorial 9 Worksheet

1) The experiment analyzed microtubule concentration and length over time above and below the critical tubulin concentration through in vitro experiments and electron microscopy. 2) Above the critical concentration, microtubule concentration remained the same while length significantly increased, indicating microtubules were dynamically growing and shrinking. 3) Below the critical concentration, microtubule concentration sharply decreased while length slightly increased, showing microtubules were mostly shrinking but still underwent some growth.

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30 views

Tutorial 9 Worksheet

1) The experiment analyzed microtubule concentration and length over time above and below the critical tubulin concentration through in vitro experiments and electron microscopy. 2) Above the critical concentration, microtubule concentration remained the same while length significantly increased, indicating microtubules were dynamically growing and shrinking. 3) Below the critical concentration, microtubule concentration sharply decreased while length slightly increased, showing microtubules were mostly shrinking but still underwent some growth.

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andrewl.0418.al
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© © All Rights Reserved
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Participation Mark ________ (1%)

BIOL200 Unit 6
Problem Solving Worksheet
Problem 6.2.8 (tags: #microtubules, #TEM, #critical concentration, #dynamic instability)
Prior to the development of live cell imaging techniques, researchers had to find creative ways to study dynamic
processes. Some of the earliest work on dynamic instability came from in vitro experiments that used electron
microscopy to analyze microtubules that had been allowed to polymerize in a test tube at different concentrations,
prior to visualization of static images and statistical analysis. The data presented here is some of the earliest work
that was done on this subject (Original Paper: Mitchison & Kirschner 1984. Nature 312: 237).

In this experiment the same concentration of short microtubules were


added to 2 separate solutions of free tubulin: in the first one, the free Figure 1
tubulin concentration was above the critical concentration, and in the
other, the tubulin concentration was below the critical concentration
(note that the critical concentration had been previously determined by
this same research group). The concentration of microtubules was
measured over time.

Figure 1 shows the number of microtubules per ml as a function of time.


Closed circles () are data points from the solution that was above
critical concentration, while open circles () are data points from the
solution that had been diluted below the critical concentration.

a) For each case, describe the change in microtubule concentration.

For the tubulin that was above the critical concentration, a slight
increase was observed at 10 mins after the dilution followed by a slight
drop under the initial concentration at 20 minutes.
For the tubulin that was below critical concentration, a steep drop of about 1-unit concentration was observed
over a time period slightly less than 5 minutes followed by another drop of about 1 unit concentration over a 5
minute period. During 10-20 minutes, a decrease of about 0.05unit concentration can be observed

b) This graph only tells you about one property of the MTs in solution (the concentration of MTs over time). List
some other properties of the MTs missing from this data.
Length
Concentration of tubulin
Rate of growing/shrinking

c) Can this data tell you why is there no change in microtubule concentration above Cc? Explain your reasoning.
No, only provides the overall number of Microtubules (stays the same). Does not tell us what is happening to the
individual MTs (growing/shrinking, etc.)

In Figure 2 (right) the researchers examined three time points shown Figure 2
in Figure 1 in more detail. They chose 500 microtubules at random
to measure using TEM, from 3 of the samples. These histograms
show a distribution of microtubule lengths at each time point. The
arrowhead at the top of the histogram identifies the mean length in
each sample. A line has been drawn (pink) from the top sample across the other 2 histograms to help you
compare.

d) For each case, describe the change in MT length.

B acts as the control


Below CC, the average length slightly increase
Above CC, significance increase in length
Above CC shows greatest variance in length
Individual MTs are able to grow regardless of [Tubulin]

e) Are all of the microtubules below the Cc behaving the same way? Justify your answer by referring to the data
from both Fig 1 and Fig 2.
No, Above CC [MT] stays the same but significantly grows in length
Below CC [MT] decreases but still slightly grows in length

f) What conclusion(s) can you draw about the relationship between microtubule concentration, microtubule
length, and free tubulin concentration? Support your conclusion by explaining what is happening to the MTs
in this experiment above and below the Cc. In your explanation, identify what data helped you come to your
conclusion(s).

Above CC
[MT] does not change, the length increases significantly as tubulin is polymerized

Below CC
[MT] decreases a lot, length of increases slightly as tubulin is depolymerized

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