Biology Reference
In-Depth Information
A
C
E
13-pf
MTs
DCX
Microtubule
TIR lasers
Axoneme
Axoneme-MTs
B
D
14-pf
Linker
S/P-rich
S/P-rich
C-DC
4
μ
m
N-DC
GMPCPP
GMPCPP-MTs
F
G
Mixed
MTs
DCX-GFP
4
μ
m
4
μ
m
4
μ
m
FIGURE 21.2
Fluorescence-based assays for microtubule architecture. (A) Schematic of the single-
molecule assay. (B) Schematic of DCX-GFP. The two DC-domains (labeled) are joined by a
linker (labeled) and flanked by polypeptides. The C-terminal polypeptide is enriched in S/P
residues (labeled). The GFP-tag is C-terminal. (C) Schematic drawing of an axoneme
(labeled), which nucleates axoneme microtubules (MTs) with
90% 13-pf. (D) Chemical
>
drawing of GMPCPP, which nucleates GMPCPP-MTs with
96% 14-pf. (E) Image of
axoneme-nucleated microtubules (white arrow) and GMPCPP microtubules in the same
microscope chamber. (F) Schematic of a mixed population of MTs nucleated from purified
tubulin. (G) Image of the mixed population of rhodamine-labeled microtubules (MTs);
image of DCX-GFP exposed to this mixed population (DCX-GFP); color-combined image
of MTs and DCX-GFP. Note that DCX-GFP binds preferentially to a subset of the mixed
population, corresponding to the 13-pf subset, and to segments within individual
microtubules (white arrow).
>
Adapted from Bechstedt and Brouhard (2012)
Therefore, adding 14-pf GMPCPP microtubules into the flow chamber provides an
important internal control for the experiment.
To polymerize the mixed microtubules, add 32
m
M tubulin, 1 mM GTP, 4 mM
MgCl
2
þ
5% DMSO and adjust with BRB80 to 12
m
l in a microcentrifuge tube. In-
cubate this mix at 37
C for 30 min. Add 200 ml BRB80
þ
10
m
M paclitaxel, pre-
warmed to 37
C, and centrifuge at 150,000
g
for 5 min (e.g., in a Beckman
Airfuge or tabletop ultracentrifuge such as a Beckman Optima MAX). Discard
the supernatant and resuspend the pellet in 200 ml BRB80
10
m
M paclitaxel.
At this point, the paclitaxel-microtubules might be too concentrated for direct use
and should be diluted up to fivefold before introduction into the flow chamber.
þ
