Biology Reference
In-Depth Information
4.3.2
GFP-tubulin expression
A more recent trend has used the expression of GFP-tubulin to label microtubules.
Although this approach eliminates many of the disadvantages of microinjection, cau-
tion again needs to be used because incorporation of this chimera can also potentially
alter the behavior of the microtubules and possibly cause their disruption leading to
problems in cell proliferation. We have found, for example, that elevated expression
of GFP-tagged
-tubulin can interfere with cell division (unpublished studies).
Thus, this approach works best when the level of expression is kept very low so that
the labeled tubulin is only present in trace amounts.
a
-or
b
4.3.3
GFP-MAP4 expression
A third method for labeling microtubules, and the one that we most commonly use,
involves the expression of GFP-labeled MAP4 (available from Life Technologies,
Grand Island, NY). MAP4 is a ubiquitous protein that binds to microtubules, but
it is not a structural component of the filaments. Thus, unlike the other approaches,
the subunit composition of the microtubules is not altered. Although this protein has
been reported to stabilize microtubules (
Nguyen et al., 1997
), we found no effects
of high overexpression on microtubule assembly, drug sensitivity, or proliferation
of CHO or HeLa cells (
Barlow, Gonzalez-Garay, West, Olmsted, & Cabral,
1994
). Similarly, another group found no phenotype when they prevented MAP4-
microtubule interactions by microinjection of antibodies into human 356 fibroblast
and monkey LLCMK2 epithelial cells (
Wang, Peloquin, Zhai, Bulinski, & Borisy,
1996
). Consistent with a lack of MAP4 effects on microtubule assembly, we
found that the residence time of MAP 4 on microtubules is very short (
5 s, unpub-
lished studies), suggesting that it is not a structural component of the filaments;
we found that measurements of dynamic instability in mammalian cells express-
ing GFP-MAP4 are nearly identical to those obtained using microinjection of
rhodamine-labeled tubulin (
Ganguly et al., 2010; Kamath et al., 2005; Yang et al.,
2010
). Ultimately, it is likely that all the methods work and give similar results pro-
vided that labeled tubulin is kept at sufficiently low levels. Because GFP-MAP4
does not incorporate into microtubules, maintaining a low level of expression is less
critical. However, keeping its expression low has the advantage that most of the pro-
tein will be associated with the microtubules, thereby providing better contrast.
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4.4
MEASURING DETACHMENT
4.4.1
Equipment
The quantification of detachment involves counting the number of such events
within a defined period of time in cells that are growing under normal or experimen-
tal conditions. To accomplish this, a reliable inverted fluorescence microscope
equipped with high power/high numerical aperture objectives, a sensitive digital
