Biology Reference
In-Depth Information
13.1.3.3
Severing and depolymerization
13.1.3.3.1
Experiment
The experiment is executed in the same way as the two-color colocalization exper-
iments except that it is only essential to use labeled microtubules. The enzyme can be
either unlabeled or labeled.
13.1.3.3.2
Analysis
13.1.3.3.2.1
Depolymerization rate analysis.
When microtubules depolymerize in
the presence of katanin, the ends shrink and depolymerization is observed as the loss
of signal at the ends of microtubules (
Fig. 13.4
A). Also, when microtubules are sev-
ered in the lattice, new ends are created and they also disassemble at these new ends.
To measure the rate of decay of the microtubule ends, create a kymograph of the
microtubule over time, use the angle tool in ImageJ to draw two intersecting lines
and measure the average angle at each end (
Fig. 13.4
C). The rate of depolymerization
is determined as one over the tangent of a given angle in distance pixel per time pixel.
We convert the rate data using the known time interval and distance scale, which de-
pends on the pixel conversion factor for the camera. Then, using graphing software,
we plot the average rate of depolymerization for the plus-ends and minus-ends of mi-
crotubules if the polarity of the microtubules is known.
13.1.3.3.2.2
Severing frequency analysis.
First, determine the length of all micro-
tubules present at the beginning of the time series of images. Then, count by hand the
number of severing events along microtubules using either the movie or the kymo-
graphs. Microtubule breaks due to severing look like an interruption in signal along
the microtubule. Sometimes, many breaks occur close together, so it is important to
be careful to count individual breaks instead of a big break made of several tiny
breaks close to each other. Given the resolution limit of fluorescence microscopy,
approximately half the wavelength of the emitted light, we require that over
200 nm of filament is removed in order to detect a break. A length of 200 nm is com-
posed of over 300 dimers. Since our microtubules are Taxol stabilized, they do not
depolymerize by having dimers removed, so the severing enzyme is most likely ac-
tively removing these dimers.
In order to quantify the density of severing events per unit time in a microtubule,
count the number of clear severs observed and divide by the length of microtubule and
the time of imaging. To quantify the frequency of severing at interfaces, use “Proto-
filament Defect Microtubules” from above (
Section 1.2.1.2.4
) and count the number of
severing events at every interface in individual microtubules. Divide by the total num-
ber of frames to acquire a severing rate. As a control, compare the frequency of sev-
ering at interfaces with the frequency of severing 1-2
m
m far away from interfaces.
13.1.3.4
Binding and diffusion
13.1.3.4.1
Experiment
1.
First, take an image of microtubules in epifluorescence.
2.
Image GFP-katanin at low concentrations using TIRF such that individual
molecules are visible. Take time-lapse movies with 100 ms frame rates, or faster,
