Biology Reference
In-Depth Information
The cells used in step 9 should be previously cultured in liquid medium to
mid-log phase.
The assembled slide will hold cells in good condition for more than 5 h of
imaging.
Materials
Glass slide 25
75
1.0 mm (Fisher Brand 12-550A3).
Coverslip 24
40-1.5 mm (Fisher Brand 12-544-C).
24.1.3
Live-cell imaging
As proof of concept, we have imaged three fission yeast strains expressing mCherry-
atb2 (tubulin): cut7.24
ts
(control), cut7.24
ts
:csi1
, and cut7.24
ts
:csi2
. csi1 and csi2
are novel fission yeast genes involved in bipolar spindle organization and chromosome
segregation (data to be published elsewhere). Deletion of either csi1 or csi2 (csi1
D
D
D
or
csi2
) results in transient monopolar spindles and subsequent chromosome segregation
defects. We hypothesize that csi1 and/or csi2 regulates mitotic spindle microtubule dy-
namics because csi1
D
D
D
also exhibit abnormally longmetaphase spindles com-
pared to wild-type cells. To test this hypothesis, we image the strains as follows:
and csi2
1.
Put the assembled slide containing cells into an incubator of 37
C for 20 min
(
Fig. 24.1
B).
At this time, cells which have begun to transit into mitosis, or cells which are still in
prophase and metaphase, will become monopolar due to the inactivation of cut7p
(
Hagan & Yanagida, 1992
). Cells which have already started anaphase will continue
through the cell cycle (
Fu et al., 2009
). As incubation at 37
C continues, more cells
will enter mitosis, resulting in more monopolar spindles.
2.
The microscope should have fluorescence imaging capability and, importantly,
have a temperature box which can maintain a stable 37
C throughout the
imaging duration.
We typically use a Yokowaga spinning disk confocal microscope equipped with a
cooled CCD- or EMCCD-camera for imaging live cells (
Tran, Paoletti, & Chang,
2004
). Our microscope is enclosed in a temperature box which stably tunes the tem-
perature from ambient (22
C) to 40
C.
3.
Quickly transfer the slide from the incubator onto the microscope which has been
preset at 37
C for live-cell imaging.
We image the cells according to the spatiotemporal requirement of the biological pro-
cess being studied. For example, individual fission yeast interphase microtubules have
an average life of
3 min (
Drummond &Cross, 2000; Tran et al., 2001
). For this time-
scale, 5-s interval sampling frequency was sufficient to capture microtubule dynamics
with high spatiotemporal resolution. In contrast, mitotic microtubules are expected to
be more dynamic. We found empirically that
individual fission yeast mitotic
