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glutamylase TTLL4, known to generate glutamylation on MTs (
van Dijk et al.,
2007
), or the deglutamylase CCP5, known to remove glutamate side chains
(
Rogowski et al., 2010
). Strikingly, at dilutions commonly used for the antibody
GT335 (0.5-0.1
m
g/ml), cytoplasmic MTs are not clearly labeled in nontransfected
cells (
Fig. 16.2
A, white contours), while all MTs are fully labeled in TTLL4-
expressing cells (
Fig. 16.2
A, green contours). In contrast, no change in modification
levels is seen in cells transfected with CCP5 with 0.5
m
g/ml GT335 (
Fig. 16.2
B,
lower panel, green contours). However, using a higher concentration of the antibody
(5
m
g/ml) reveals a clear MT staining in nontransfected U2OS cells (
Fig. 16.2
B, up-
per panel, white contours), while this staining is strongly decreased in cells expres-
sing the deglutamylase CCP5 (
Fig. 16.2
B, upper panel, green contours).
This demonstrates that U2OS cells carry low levels of glutamylation on their in-
terphase MTs (
Regnard, Desbruyeres, Denoulet, & Edd
ยด
, 1999
), which can only be
revealed at higher concentrations of GT335 antibody. This antibody concentration is,
of course, too elevated for the detection of MTs with higher levels of PTMs, where
evenmuch lower concentrations of the same antibody are sufficient for strong labeling
(
Fig. 16.2
A, lower panel). While this conclusion might appear trivial in this overex-
pres
sion experiments, it is important to point out that similar situations might be en-
countered in cells. For instance, singleMTs in cellsmight acquire specific functions by
slight changes in MT PTMs, which could be easily overseen using antibodies at high
dilutions. Conversely, in cells containing MTs with very high modification levels,
such as neurons, developmental changes in MT PTM patterns can only be observed
if antibodies are sufficiently diluted. To illustrate this notion, we show here the stain-
ing of a 3-day-old rat cortical neuron, stained with 0.1 mg/ml of GT335. Despite the
fact that MTs in young neurons are not fully glutamylated (
Audebert et al., 1994
;
Fig. 16.2
C), and despite the high dilution of the antibody, the MTs in these neurons
appear already strongly labeled, suggesting that even higher dilutions of GT335might
be required to determine changes inMT glutamylation during neuronal development.
Finally, it should be noted that not only the density of the tubulin molecules
within an MT but also the density of MTs within certain cellular structures, such
as neuronal axons and dendrites (
Fig. 16.2
C), axonemes in cilia and flagella
(
Bressac et al., 1995
), or other MT bundles in cells, such as midbodies in anaphase
cells (
Fig. 16.1
), preclude the possibility of identifying specific PTM identities of
single MTs within these MT arrays. Because there is as yet no easy approach to cir-
cumvent this problem (even super resolution light microscopy is not able to fully
resolve single MTs in dense bundles), it should be taken into account when interpret-
ing immunostaining results from fixed cells.
16.1.3
Antibody and sample concentration in immunoblot analysis
Strikingly, tubulin concentration is also a critical factor in immunoblot analyses, es-
pecially if semi-quantitative information about the levels of PTMs should be deduced
from the detection levels with the different antibodies. Apart from the above-
discussed importance of using a range of dilutions of the antibodies to determine
