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a wide dynamic range to effectively capture much less intense single microtubules in
a single image. These limitations vary considerably among different cell lines mak-
ing some of them more optimal than others for detecting microtubule detachment
events. Ideal cell lines should be thin and well spread, and they should have a cen-
trosomal area that is populated with relatively few microtubules. We have had good
experience using Chinese hamster ovary (CHO) and vascular endothelial cells from
human umbilical cord (HUVEC) for these measurements.
One of the first published studies describing microtubule detachment involved
PtK1 cells, a very flat cell line, but one with a relatively high microtubule density
( Keating, Peloquin, Rodionov, Momcilovic, & Borisy, 1997 ). To optimize the visu-
alization of detachment events, the authors recommended using cells in which the
centrosome is situated on the ventral surface with microtubules that run under the
nucleus, a suggestion that we have adopted. Using cells with this orientation effec-
tively reduces the three-dimensional space to two dimensions while also providing
better contrast for image capture ( Fig. 4.1 ).
4.2 MICROTUBULE DETACHMENT IN SPINDLES
Mitotic cells present an especially difficult challenge because they are frequently
round, lack a nucleus, and have two spindle poles rather than a single centrosome
nucleating microtubules. Mitotic cells are also thicker than interphase cells thus
causing poor contrast and difficulty tracking individual microtubules in the three-
dimensional space. Other cells such as PtK2 remain well attached during mitosis
but are still difficult to image because of the high density of microtubules in the
FIGURE 4.1
CHO cells transfected with EGFP-MAP4. Panel A shows an interphase cell with
the centrosome (marked with an asterisk) on the bottom (ventral) surface and microtubules
clearly visible under the dark nuclear area. Panel B shows a cell in the same dish with
the centrosome near the upper surface. Microtubule density and overall fluorescence intensity
are too high to detect individual microtubules at the centrosome in panel B. Scale bar
5
m
m.
ΒΌ
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