Assembly promoting activity of CLIP-170 fragments.
A–C, MT assembly in the presence of CLIP-170 fragments was
monitored by change in the absorbance at 350 nm. Polymerization of tubulin (12
μm) was measured in the absence and presence of the different
CLIP-170 fragments (2.0 μm) as shown. In this assay,
H1-(1–350) (2.0 μm) causes strong bundling and thus
produces an off-scale light scattering signal (A, asterisk). To
minimize this effect and for comparisons with the other CLIP-170 fragments,
H1-(1–350) was also used at 1.0 μm as shown. Data were
extracted at different time points and plotted using the GraFit 6 program; the
lines are provided as a guide. Each data point is the average from
two independent experiments. D–F, polymerization with or
without CLIP-170 fragments as shown was carried out in the presence of taxol
seeds (1.0 μm) to observe the effects on microtubule elongation.
The tubulin concentration was 12 μm and all CLIP-170 fragments
were used at 1.0 μm concentration to minimize possible bundling
effects.