Biology Reference
In-Depth Information
10.3.2.6.2
Attachment of microtubule seeds and elongation
Microtubule seed suspension is diluted 10 times in PEM containing 10
m
MTaxol and
100
m
M ATP. Once microtubule seeds are loaded, the chamber is placed at 37
Con
a Zeiss LSM510 confocal microscope (Heidelberg Germany). Microtubule seed at-
tachment and movement are monitored by acquisition of the Cy5 fluorescent signal
(63
1.4 NA objective).
Seed gliding on kinesin-coated glass allows the unambiguous identification of
microtubule plus and minus ends: because immobilized kinesin walks toward the
plus ends, the seeds move with minus ends in front. To stop seeds from gliding prior
to elongation, the chamber is washed twice with PEM-C supplemented with 10
m
M
Taxol and 1 mM AMPPNP.
To elongate microtubule seeds, a 1 g/L mix of unlabeled and Cy3-labeled tubulin
(with a 1:4 ratio of labeled to unlabeled tubulin) in PEM supplemented with 1 mM
GTP is perfused twice in the chamber to eliminate Taxol and to avoid stabilization of
growing microtubules. To monitor microtubule elongation, time-lapse sequences are
acquired during 20 min. Microtubules that elongate at the plus and minus ends of the
seeds interact with the kinesin adsorbed on glass and polymerize parallel to the con-
focal plane. At the end of microtubule growth, free tubulin is removed by washing
the chamber with PEM-G containing 1
m
M Taxol.
Tips
- Imaging of microtubule polymerization is done at 37
C. Placing a humid lid
over the incubation chamber so that liquid in the channel does not evaporate
from its extremities is strongly recommended.
- We also recommend using an oxygen scavenger cocktail (20 mM
D
-glucose, 0.02 mg/mL glucose oxidase, 0.08 mg/mL catalase, and 0.5%
b
-mercaptoethanol). Oxygen scavenging protects dyes from
photobleaching and prevents tubulin oxidation, which can cause the
spontaneous breaking and depolymerization of microtubules. This cocktail
is added to every solution injected in the chamber before imaging. It has to
be used within 1 h after preparation.
To label GTP-tubulin, we use the protocol described above (see
Section 1.2
). After
labeling, microtubules can be imaged directly or fixed with methanol for further
analysis (
Fig. 10.3
).
10.4
DISCUSSION AND FUTURE PROSPECTS
The MB11 conformational antibody specifically binds to GTP-tubulin. It is a unique
tool to study the distribution of GTP-tubulin islands along microtubules and their
implication in microtubule dynamics. Although the exact structure that the antibody
recognizes in tubulin is not yet known, MB11 may prove to be a powerful tool to
study modifications in the microtubule network depending on changes in the cell
status in pathophysiological conditions (e.g., differentiation, division, migration).
