Biology Reference
In-Depth Information
microtubules have an average life time of less than 1 min. This short timescale requires
at least 2-s interval sampling frequency to sufficiently capture mitotic microtubule
dynamics.
Notes
For step 1, the slide can be placed directly onto the microscope stage set at
37
C for 20 min prior to imaging. This would negate the need for an
incubator.
During the transfer of the slide from the incubator to the microscope, it is
critical that the transfer time is less than 1 min. cut7.24
ts
is a fast acting
mutant (
Velve Casquillas et al., 2011
). Within
1 min at the
nonpermissive temperature (37
C), cut7p is inactive; and within
1 min
at the permissive temperature (25
C), cut7p is active again. To ensure
sustained monopolar spindles, we recommend that the transfer time is
completed under 30 s.
Ultimately, the practical achievable spatiotemporal resolutions of imaging
individual spindle microtubules will depend on many factors: (1) the
quality and speed of the optical pathway, (2) the sensitivity of the detector
of the microscope, (3) the exposure time needed to acquire a good
fluorescent signal, (4) the bleaching of the fluorescent signal, etc.
Compromises will be necessary to achieve optimal results.
Materials
37
C incubator.
Optical microscope capable of fluorescent imaging and has a temperature
control box.
24.1.4
Data analysis
Figure 24.1
C shows two examples of the “control” cut7.24
ts
strain expressing
mCherry-atb2 (tubulin). cut7.24
ts
is reversible, able to transform from a short bipo-
lar spindle (represented by the “bar” of fluorescent signal) to a monopolar spindle
(represented by the “aster” of microtubule protrusions) at the nonpermissive tem-
perature, and then back to a bipolar spindle when the temperature is again permis-
sive. This is an important control to show that the inactivation of cut7p is reversible
and does not permanently alter spindle dynamics and organization, strongly imply-
ing that individual microtubule dynamics is not affected by the inactivation of
cut7p. Therefore, cut7.24
ts
is a good tool to study individual spindle microtubule
dynamics.
Figure 24.1
D shows comparisons of spindle morphology between the wild-type
and control cut7.24
ts
, and the mutants cut7.24
ts
:csi1
and cut7.24
ts
:csi2
D
D
at permis-
sive and nonpermissive temperatures. The wild-type spindles remain bipolar at both
temperatures. The control cut7.24
ts
bipolar spindles become monopolar at the non-
permissive temperature. Both mutants cut7.24
ts
:csi1
and cut7.24
ts
:csi2
have tran-
sient monopolar spindle phenotype even at the permissive temperature as part of the
D
D
