Biology Reference
In-Depth Information
FIGURE 4.2
Microtubule detachment. An early prophase CHO cell expressing EGFP-MAP4 is shown in
successive frames taken 5 s apart. A detaching microtubule traced with a dotted line (or
colored red in the online version) is seen to shorten at its minus end (arrow, panel B). Only one
spindle pole is in the plane of focus. Scale bar
5
m
m.
ΒΌ
adjusting the focus knob while taking images of the cell with brief illumination. Once
in good focus, images are taken every 5 s for 55 frames. More frequent sampling may
cause bleaching of the fluorescence as well as cell damage depending on exposure
times and intensity of the light source. Image stacks may be deconvolved, if neces-
sary, to improve contrast.
Detachments are detected by examining successive frames in each stack for a
clear release of one or more microtubules from the centrosome region (e.g., see
Fig. 4.2
). Microtubules with free ends near the centrosome that suddenly appear
in a frame and were not clearly attached in the previous frame are not counted be-
cause these may have moved into the plane of focus by chance, and it is often difficult
to establish whether the free end is the plus or minus end. Any released microtubules
are observed for several more successive frames to determine whether they exhibit
the typical behavior of growth and shortening of the plus end (when visible), short-
ening (but not growth) from the minus end, and translocation away from the centro-
some. To minimize drift, manual adjustments are made during image capture to keep
cellular markers such as intersections between microtubules in sharp focus.
We generally accumulate data for 10-20 cells in this way and quantify the results
as the number of detachment events that we see divided by the total recording time
examined. For wild-type CHO cells, we typically see a detachment rate of 0.17/cell/
min during interphase and a rate seven times as high in prophase cells (
Yang et al.,
2010
). Other published rates have varied from 0.02 to 1.5/cell/min during interphase
(
Keating et al., 1997; Waterman-Storer & Salmon, 1997
). Some of these differences
may arise from the use of different cell lines, but there are also likely to be differences
