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changing the gene expression profiles, get incorporated into newly formed MTs.
Thus, tubulin PTMs, which include acetylation, detyrosination, polyglutamylation,
and polyglycylation, are the prime candidates for fine-tuning MT functions
(
Box 16.1
; reviews:
Janke & Bulinski, 2011; Luduena, 2013
).
Despite the variety of functions tubulin PTMs are expected to fulfill, current re-
search in the MT field often fails to correctly assess the importance or even the pres-
ence of these modifications. Functional studies have been difficult because most of
the enzymes catalyzing tubulin PTMs had not been discovered until recently (
Akella
et al., 2010; Janke et al., 2005; Kimura et al., 2010; Rogowski et al., 2009, 2010;
Shida, Cueva, Xu, Goodman, & Nachury, 2010; van Dijk et al., 2007; Wloga
et al., 2009
). Moreover, tubulin PTMs are hard to quantify or even detect. For in-
stance, glutamylated peptides are often not detected in classical mass spectrometry,
a problem that has been overcome for purified tubulin, but that requires relatively
large amounts of protein (
Redeker, 2010
). Furthermore, modifications do not signif-
icantly alter the migration properties of tubulin on a sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE); hence, gel-shift cannot be used to
assay these modifications.
Thus, the most reliable way for distinguishing differentially modifiedMTs and tu-
bulin in tissues, cells, and by immunoblot is PTM-specific antibodies. While some of
these antibodies have proven to be excellent tools, precise data about their specificities
and limitations have not been comprehensively reported. Here, we present a collection
of well-analyzed antibodies specific to tubulin PTMs, together with appropriate pro-
tocols that allow using them in a semi-quantitative fashion in immunofluorescence on
cells, as well as in immunoblot. We illustrate the opportunities and limitations of the
described methods based on the example of glutamylation-specific antibodies.
16.1
OBSERVATIONS
16.1.1
Impact of cell fixation on antibody labeling
We have found that the cell fixationmethod is crucial for the correct preservation of the
MT cytoskeleton, as well as for the quality of detectionwith some of themodification-
specific antibodies. Neither a paraformaldehyde (PFA)-sucrose fixation nor the fix-
ation with cold methanol provides satisfying results, especially for the staining of MT
modifications of certain structures, as for instance the midbody. Therefore, we use a
method in which the cytoskeleton is prefixed with a bifunctional protein cross-linker,
dithiobis succinimidyl propionate (DSP;
Bell &Safiejko-Mroczka, 1995
). To demon-
strate the impact of these three methods on immunolabeling of cultured cells, we have
fixed HeLa cells that were cultured on fibronectin-coated coverslips and stained the
cells with polyE and 12G10 antibodies. Cells in anaphase, representing a midbody
structure, were observed (
Fig. 16.1
).
Using the DSP fixation method, we have previously shown that midbody MTs
carry long glutamate side chains (
Lacroix et al., 2010
); however, this specific label-
ing is only seen when cells are fixed with the DSP-PFA method. In contrast, fixing
