Biology Reference
In-Depth Information
Side view
B
Coverslip
Cells
Agar 2%
PDMS
Glass slide
Cell culture
and
slide assembly
20 min @ 37
°
C
to insure cut7
is completely
inactivated
Imaging:
-Z-Stacks
-every 2 s for
high temporal resolution
37
Top view
25
Coverslip
Cells
Agar 2%
PDMS
Glass slide
0
20
Time (min)
C
D
25
C
37
C
cut7.24
ts
mCh-atb2
0
25
C
4
37
C
13
25
C
25
FIGURE 24.1
Amethod to image individual mitotic spindle microtubules in fission yeast. (A) Schematic view
of the specimen slide assembly. (B) Schematic view of the temperature shift experiment. Cell
culture and slide assembly are performed at ambient room temperature (25
C) prior to
shifting for 20 min to the nonpermissive temperature (37
C) and then transferred to the
microscope set at 37
C for imaging. (C) Control cut7.24
ts
cells expressing mCherry-atb2
(tubulin) under permissive, nonpermissive, and then permissive temperatures. Z-stacks
(4 sections, 1
m spacing) are captured at 1-min intervals. Shown are maximum projection
time-lapse images. cut7.24
ts
cells can produce reversible monopolar spindles. Bar: 2
m
m.
(D) Comparisons of spindle morphology between the wild-type and control cu7.24
ts
, and the
mutants cut7.24
ts
:csi1
m
and cut7.24
ts
:csi2
expressing mCherry-atb2 at permissive and
nonpermissive temperatures. Z-stacks (4 sections, 0.5
D
D
m spacing) are captured at one time
point. Shown are maximum projection time-lapse images. Dashed yellow outlines show
cells with normal bipolar spindles. Dashed red outlines show cells with monopolar spindles.
Zoom in shows 5
m
magnification of the different spindles. Bar: 2
m
m.
