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the same cells with either the PFA or the cold methanol method prevents the detection
of this polyglutamylated MT population (
Fig. 16.1
), indicating that polyE is sensitive
to the fixation method. In contrast, in all three fixation methods, the tubulin antibody
12G10 shows a niceMT labeling (
Fig. 16.1
), which can be particularly misleading as it
suggests that MTs have been fully preserved. A possible explanation for the divergence
between polyE and 12G10 labeling on midbody MTs is the assumed low level of poly-
glutamylation on those MTs. Partial disassembly of MTs during PFA and methanol
fixations would affect rare epitopes much more than the abundant (and anyway too
dense—see
Section 16.1.2
) epitopes of tubulin. Though this effect has mostly been
observed with polyE antibody, it is likely that other modification-specific antibodies
are also sensitive to the cell fixation. We therefore strongly recommend testing differ-
ent fixation conditions if specific PTMs of MTs are tested, even if the MTs are appar-
ently intact if detected with a PTM-independent antibody, or with an antibody for a
tubulin PTM that is highly enriched on the specific MTs investigated.
16.1.2
The importance of antibody concentration in analyzing
MT PTMs in cells
A MT is a hollow tube of 25 nm of external diameter and is in most organisms com-
posed of 13 protofilaments. Each protofilament is a linear assembly of
a
-/
b
-tubulin
dimers, and protofilaments assemble side by side to form the MTs. Considering that
the diameter of an
a
-or
b
-tubulin molecule is about 4-5 nm, the dense assembly of
MTs generates a situation where similar epitopes are arranged in a distance of about
4 nm. An average antibody has an approximate diameter of 15 nm length and width,
a size that is even further increased by the binding of secondary antibodies that can
take the space of a cube, the side length of which could be around 30 nm or more.
Thus, it is obvious that the spatial constraints on the MT will not allow for a stoichio-
metric decoration of epitopes on each tubulin molecule. In other words, only a frac-
tion of the tubulin molecules present in the MT lattice can be labeled with antibodies
due to space constrains. This creates a severe problem when antibodies directed
against PTMs of tubulin are used to estimate the modification status and level of
a given MT population using immunolabeling in fixed cells.
While it is easy to distinguish MTs carrying very low levels of modification from
those with very high levels in cells, it is much harder to see subtle differences, which
could still be highly relevant from a functional point of view. Moreover, even in sit-
uations where only a given percentage of the tubulin molecules in the MT lattice are
posttranslationally modified, decoration with modification-specific antibodies could
generate strong and virtually continuous signals that cannot be distinguished from
those obtained from MTs with much higher modification levels. To avoid misinter-
pretations of the modification status of MTs based on antibody labeling, it is essential
to test serial dilutions for each of the antibodies used. Moreover, it is important to
perform such tests for each novel experimental setting.
To illustrate the importance of dilution in the correct assessment of changes in
tubulin modifications, we have transfected the U2OS cell line with either the