Biology Reference
In-Depth Information
used as an alternative to axonemes, as the
g
-tubulin ring complexes also provide a
stable, 13-pf nucleation template (
Evans, Mitchison, & Kirschner, 1985
).
Dilute the purified axoneme stock (e.g., 1:4000) in BRB80 such that only a few ax-
onemes appear in each field of view. Introduce diluted axonemes into a microscope
chamber together with an anti-TAMRA antibody (1:100 in BRB80) and incubate for
5 min. Both the axonemes and the antibody adhere nonspecifically to the cover glass
surfaces. Rinse the chamber with BRB80. Incubate the chamber with BRB80
1%
Pluronic F-127 for 5-60 min to passivate the surfaces. Rinse four times with BRB80.
Place the chamber onto themicroscope with the objective heater set to 35
C. Introduce
polymerization buffer and incubate for
þ
10 min to elongate microtubules from the ax-
oneme templates. Progress can bemonitored by taking images in the tubulin color chan-
nel. When the microtubules are 10-20
m
m in length (or as desired), rinse the chamber
with BRB80
10
m
M paclitaxel to stabilize the newly formed microtubules. Take an
image to record the location of the 13-pf axonemal microtubules.
While preparing the 13-pf axonemal microtubules, also prepare 14-pf GMPCPP
microtubules. To polymerize GMPCPP microtubules, add 2
m
M TAMRA-labeled
tubulin, 1 mM GMPCPP, and 1 mM MgCl
2
and adjust with BRB80 to 50
m
lina
microcentrifuge tube. Incubate the mixture on ice for
þ
10 min. Polymerize the
microtubules by incubating at 37
C for 2 h. Add 200 ml BRB80, prewarmed to
37
C. Centrifuge at 150,000
g
for 5 min (e.g., in a Beckman Airfuge or tabletop
ultracentrifuge). Discard the supernatant and resuspend the pellet by gentle pipetting
in 200 ml BRB80
paclitaxel. GMPCPP-stabilized microtubules depolymerize very
slowly and should be used on the same day.
After recording an image of the 13-pf axonemal microtubules, the TAMRA-
labeled GMPCPP microtubules diluted into BRB80
þ
paclitaxel can be introduced
into the flow chamber. The GMPCPP microtubules bind to the chamber surfaces
via the anti-TAMRA antibodies introduced prior to the axonemes, creating a micro-
scope chamber containing both axonemal microtubules and GMPCPP microtubules
(
Fig. 21.2
). At this point, fluorescent MAPs of interest can be introduced, and pref-
erential binding to either the axonemal microtubules or the GMPCPP microtubules
can be detected (
Bechstedt & Brouhard, 2012
).
þ
21.2.4
Comparison of mixed populations of microtubules with an
internal 14-pf control
Microtubules nucleated in the presence of GTP will range from 11-pf to 16-pf (
Sui &
Downing, 2010
). The ratio of the different pf species varies within the literature, but
the consensus is that the fraction of 13-pf microtubules is between 35% (
Moores
et al., 2004; Ray et al., 1993
) and 75% (
Vitre et al., 2008
), with the remainder con-
sisting primarily of 14-pf. The larger and smaller species are a small minority. Mixed
populations are a useful tool, as variations due to the nucleation conditions, stabiliz-
ing agent, tubulin preparation, fluorescent labeling, etc., are minimized. In our hands,
however, day-to-day variability in the ratio of 13-pf microtubules is commonplace.
