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preferentially to GTP microtubules. The authors polymerized chimeric microtubules
composed of a GDP segment and GMPCPP ends and found that kinetochores display
a higher affinity formicrotubules plus ends. This suggested that structural features of the
GTP-bound microtubule lattice could be recognizable. In the same study, it was con-
firmed that Taxol binding to tubulin mimics the GTP-bound conformation of tubulin
dimers as previously showed by
Arnal and Wade (1995)
. Indeed, kinetochores bind
equally to GMPCPP or GDP portions of microtubules polymerized in the presence
of Taxol. In a more recent example,
Maurer, Bieling, Cope, Hoenger, and Surrey
(2011)
showed that EB1, a plus end-tracking protein, andMal3, its homologue in yeast,
bind effectively toGTP- andGTP
g
S-tubulin, but onlyweakly toGMPCPP seeds and to
the GDP lattice of microtubules polymerized
in vitro
.
Such biochemical studies of the GTP cap were done
in vitro
, but
in vivo
studies
have long been missing. To study the GTP cap in cells, we selected a recombinant
antibody (MB11) using biotinylated GTP
g
S-tubulin as an antigen (
Dimitrov et al.,
2008
). Using cosedimentation experiments or immunostaining of
in vitro
polymer-
ized microtubules, MB11 was shown to recognize almost exclusively GTP-loaded
microtubules (
Dimitrov et al., 2008
). Whether MB11 directly recognizes a parti-
cula
r conformation displayed by GTP-tubulin in the polymer (like EB or CLIP170
proteins do) or whether it recognizes some structural defects is still debated. MB11
efficiently stains the plus end of microtubules that are polymerizing and thus allowed
to confirm the existence of the GTP cap at the plus ends of growing microtubules in
cells. In addition, it led us to propose that GTP-tubulin is present not only at the plus
ends but also in discrete dots along the microtubule lattice. We showed in cells that
these GTP-tubulin islands are sites of rescue events. We thus proposed that GTP hy-
drolysis in microtubules might not always be complete and that, upon microtubule
depolymerization, a GTP-tubulin island becomes exposed at the plus end and may
behave as a GTP cap to promote a rescue event. The mechanisms of rescue involving
GTP islands and the mechanisms of catastrophe are summarized and discussed in a
recent review (
Gardner, Zanic, &Howard, 2013
), which points out the importance of
rescues in the regulation of microtubule interactions with the cell cortex or with
kinetochores.
Studying GTP-tubulin conformation in microtubules using the MB11 antibody is
a technical challenge because the MB11 antibody does not recognize GTP-tubulin in
microtubules after fixation, probably because fixation alters microtubule conforma-
tion. In this chapter, we present four protocols to study the GTP cap at microtubule
plus ends and the GTP islands along the microtubule lattice using the anti-GTP-
tubulin antibody MB11 in permeabilized cells and in
in vitro
assays.
10.1
IMAGING GTP ISLANDS IN PERMEABILIZED CELLS
MB11 is a conformational antibody. As such, it is very sensitive to antigen denatur-
ation and the best staining results are obtained in permeabilized cells without fixa-
tion. To preserve the whole microtubule network, cells should be permeabilized
