Biology Reference
In-Depth Information
- The supernatants over pelleted microtubules must be taken off with great
care to avoid losing microtubules at each cycle of centrifugation between
labelings and washes.
10.3.2
Microtubules elongated from Taxol seeds immobilized on
kinesin-coated glass
10.3.2.1
Preparation of MAP-free tubulin
MAP-free tubulin is purified from porcine brain as described previously by
Walker et al. (1988)
. MAPs are removed by phosphocellulose chromatography
and tubulin aliquots are frozen in liquid nitrogen and stored at
80
C. For each ex-
periment, an aliquot of purified tubulin is thawed on ice and centrifuged at 20,000
g
for 20 min to sediment aggregated tubulin, which is discarded.
10.3.2.2
Fluorescent-tubulin labeling
Tubulin is labeled with Cy3 or Cy5 monoreactive NHS ester dyes using a protocol
adapted from
Peloquin, Komarova, and Borisy (2005)
. For one labeling, 500
m
gof
MAP-free tubulin (10
m
g/
m
L) is polymerized with 1 mM GTP for 30 min at 37
C.
One vial of Cy3 or Cy5 monoreactive dye is solubilized in 20
m
L DMSO and
polymerized microtubules are incubated for 15 min at 37
C with 5
m
L of this solu-
tion. Microtubules are then depolymerized for 10 min on ice and labeled tubulin is
separated from the excess of unconjugated dye by two cycles of assembly-disassem-
bly. Tubulin-labeled aliquots are frozen in liquid nitrogen and stored at
80
C.
The advantage of this protocol is that tubulin is labeled in polymerized microtu-
bules and dyes cannot react with primary amines involved in the interaction between
tubulin heterodimers during microtubule polymerization. Thus, labeled tubulin is not
excluded from microtubules when combined with unlabeled tubulin in the polymer-
ization protocols described above. We could not exclude however that tubulin is la-
beled on sites important for MAP interactions. In polymerization studies, we used
less than 25% of labeled tubulin to avoid the inhibition of MAP interactions with
microtubules due to the presence of the fluorochrome.
10.3.2.3
Recombinant KHC reconstitution
In these experiments, microtubules are specifically bound on glass coverslips using
the interaction with recombinant, active KHC. We use a protocol described for mi-
crotubule gliding motility assays (
Howard, Hunt, & Baek, 1993
), with minor mod-
ifications. Classical gliding assay using ATP is first used to determine the polarity of
microtubule seeds. Then, microtubule growth is monitored in the absence of move-
ment because of kinesin loading with the nonhydrolyzable ATP analogue, AMPPNP,
which allows microtubule/kinesin interaction but prevents nucleotide hydrolysis
necessary for microtubule displacement.
