Oscillations, Waves, and Interactions - GWDG

448 S. Lakämper and C. F. Schmidt
Figure 11. Single-molecule binding events generated by full-length ncd in a three-bead
assay. (a) Raw data, 2-kHz sample rate, immobilized ncd interacting with a suspended
microtubule in the presence of 2 µM ATP. Displacements are measured parallel to the long
axis of the microtubule and plotted against time. (b) Standard deviation of the raw data in
(a), calculated using a 50-point (1-ms) moving window. (c) Wavelet-filtered s.d. data. The
beginning and end of each event were determined by thresholding the wavelet-filtered data
(dashed line; from Ref. [16]).
class of tetrameric kinesins, the Kinesin-5s, in particular the Eg5 motor of Xenopus
laevis [9,10,18]. Eg5 has two pairs of motor-domains at each end of an extended
tetrameric coiled-coil. Its cellular function is to aid in the morphogenesis of bipolar
mitotic spindle during cell division. We could show that Eg5 dynamically crosslinks
microtubules and thus provides the forces necessary to slide the spindle poles apart.
We used an in vitro assay with purified Eg5 and fluorescently labeled microtubules.
We bound bundles of microtubules (axonemes) to a glass coverslip, added motors and
more microtubules. We found that single microtubules readily bound to and aligned
with axonemes in the presence of Eg5 [18]. Approximately half of the microtubules
were immotile or moved very slowly (< 10 nm/s) whereas the rest moved along the
axonemes with an average velocity of 40 nm/s. With polarity marked microtubules
as both tracks and substrate we could prove that only anti-parallel microtubules
displayed relative motility. The relative movement of microtubules that were not
aligned parallel showed that the motors could move simultaneously with respect to
both linked microtubules (Fig. 12, Ref. [18]).
While these assays clearly indicated the capability of Eg5-kinesin to cross-linking
and driving anti-parallel microtubules it was not clear whether the motility was driven
by single Eg5-tetramers or by functional aggregates or patches of Eg5 at the cross-link