Biology Reference
In-Depth Information
2.
Establish the best exposure time for the microtubules. We typically take time-
lapse movies with 5-20 s between frames.
3.
Perform a control experiment without severing enzymes to test that buffers
and imaging conditions do not cause microtubule break down. Create a
chamber with microtubules, record for 3 min, and then flow through with
imaging buffer. Add the imaging buffer slowly and carefully so that you do
not change the position or lose focus (we use the Perfect Focus System of our
Ti-E microscope). If the microtubules go out of focus, stop recording and
refocus before beginning to record again. Record a 20-min movie to
determine that the microtubules are not photodamaged due to imaging alone.
If photodamage occurs, readjust the imaging parameters (exposure time or
time between frames) so that you are not damaging the microtubules.
4.
To test that the severing enzyme is functional, repeat the same experimental
procedure as with the control chamber, except include severing enzyme in the
imaging buffer that contains the ATP and oxygen scavenging system. Begin with
a low concentration (50 nM) and work up to higher concentrations to find the
concentration that enables active severing.
13.1.3.2
Two-color colocalization
13.1.3.2.1
Experimental procedures
For two-color imaging, we perform the same experiment described above, except
we set up the microscope to record sequential images in red and green fluores-
cence. Otherwise a dual-view imaging system can be employed for simultaneous
imaging.
13.1.3.2.2
Analysis
1.
The data are recorded as 16-bit images using NIS Elements AR software (Nikon).
The data are exported at 16-bit tiff files for each color.
2.
All movies were aligned using the ImageJ plug-in stackreg in translation mode
(
Th´venaz, Ruttimann, & Unser, 1998
).
3.
The motion of single GFP-katanin molecules and the fluorescence signal
of microtubules are analyzed using kymographs (space-time plots)
generated with the Multiple Kymograph plug-in for ImageJ (J. Rietdorf
and A. Seitz, European Molecular Biology Laboratory, Heidelberg,
Germany). The kymographs are graphical representations of spatial
position over time in which the
y
-axis represents the progression of time as
going downward (
Fig. 13.4
). To generate kymographs from a stack of
tiffs, draw a segmented line on each microtubule. Restore the selected line
on the green channel stack of images. Use the kymograph plug-in on both
channels using the same selected microtubule region of interest.
Kymographs are a useful tool for looking at both the microtubules and the
labeled severing enzyme because you can evaluate when the enzyme is found
at a severing event.
