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FIGURE 10.3
GTP-tubulin labeling in microtubules elongated from Taxol-stabilized seeds attached to
kinesin heavy chain. Taxol-stabilized microtubule seeds prepared using Cy5-labeled tubulin
were attached to kinesin. After seed immobilization with AMPPNP, microtubules were
elongated with Cy3-labeled tubulin and then subjected to MB11 staining revealed with
A488-labeled secondary antibody. Fields from two experiments are shown. Images are
confocal planes acquired with a Zeiss LSM510microscope. GTP caps and internal GTP islands
are indicated with arrows and arrowheads, respectively. Scale bar
10 mm. Signals have been
artificially increased for better visualization in the black and white version of the figure.
¼
Certain microtubule-stabilizing MAPs, like the Von Hippel-Lindau antioncogene
( Thoma et al., 2010 ), may modulate the occurrence of microtubule internal GTP
islands. It suggests that cells may finely regulate rescue events by controlling the
local density of GTP islands. Understanding how GTP islands occur in microtubules
and if other MAPs facilitate their persistence is an important challenge. Conversely,
understanding whether GTP-bound tubulin in the internal regions of microtubules or
in theGTP cap of growingmicrotubules can be readily recognized by
TIPs to control
microtubule elongation and rescues will be of great interest to get a more comprehen-
sive view of rescue events. However, colabelings of GTP-tubulin and þ TIPs in living
cells are still an open challenge. Gaining access to such information will be valuable,
for example, to measure the actual size of the GTP cap or to evaluate whether
GTP-tubulin conformation is immobile or can propagate along microtubules.
þ
Acknowledgments
We thank A. Dimitrov (Institut Curie, Paris) who contributed to the setup of some of the pro-
tocols described here. We thank L. Sengmanivong for support in SIM microscopy and
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