Biology Reference
In-Depth Information
with no delay between frames. Assays can be performed with the enzyme in
trapped kinetic states using AMPPNP to mimic ATP state, ATP, ADP, or
hexokinase to mimic Apo (no-nucleotide) state. The experiments elucidate the
effect of the nucleotide on the binding and motility of the microtubule-severing
enzyme.
13.1.3.4.2
Analysis
1.
The quantitative measurement of binding of GFP-katanin is determined by
counting the number of binding events manually.
2.
The duration of association for each event is measured from kymographs by
recording the number of pixels (frames) each molecule is bound. The molecule
must associate and dissociate within the movie to be included. We do not include
molecules that are already associated before the start of the movie. The number
of pixels needs to be converted to seconds using the frame rate, typically
100 ms/frame.
3.
Diffusion is determined by tracking individual and well-separated fluorescent
enzymes using the SpotTracker plug-in in ImageJ. First, rotate the frames of the
movie so that the microtubule is horizontal. This creates diffusion in one
dimension and is easier to analyze.
4.
Run the SpotTracker plug-in and save the results.
5.
Analyze the results in Excel by calculating the mean squared displacement and
plotting it over elapsed time shift,
Dt
, as follows. For each time shift of
Dt
¼
1, 2,
3,
x
N
)
as
Dx
i
, where
i
represents the index of the displacement. For instance, for a frame
shift of one frame,
Dt
is 1, and one calculates
Dx
1
¼
...
,
N
frames, calculate the difference between two positions (
x
1
,
x
2
,
x
3
,
...
x
2
x
1
,
Dx
2
¼
x
3
x
2
,
Dx
3
¼
x
4
x
3
,
...
. This is the “displacement” part of the mean-squared
displacement.
6.
Next, you must square these displacements:
Dx
i
2
.
7.
Finally, the squares of the displacements must be averaged for each time shift so
that you get the average over all
i
of
Dx
i
2
for each time shift,
Dt
.
8.
Finally, plot the averages of the squared displacements as a function of
Dt
. Fit a
line to the resulting data. The slope equals the displacement squared per unit
time, which is a diffusion coefficient. Make sure to convert the data from pixels to
m
m
2
/s. See
Table 13.1
for an example data set and the plot is featured in
Fig. 13.4
.
13.1.3.5
Photobleaching
13.1.3.5.1
Experiment
To quantify the oligomeric state of GFP-katanin, we used photobleaching assays.
These assays can be performed by flowing in 0.01 mg/ml anti-GFP antibodies in
a flow chamber to fix GFP-katanin on the surface of the coverslip. After incubation,
we flow GFP-katanin with a nucleotide to test the oligomerization state in the pres-
ence of ATP, or AMPPNP. Then, we image a field with many spots continuously
with the laser until they become dark.
