Oscillations, Waves, and Interactions - GWDG

DPI60plus – a future with biophysics 447
Figure 10. Schematic representation of the three bead-assay used for the determination of
non-processive motor interactions (from Ref. [16]).
3.2 The chemo-mechanical cycle of Kinesin-1
According to this model, Kinesin-1 dimers in solution have ADP bound and can
only bind weakly to microtubules. When a motor interacts with the microtubule,
one of the two heads rapidly looses the ADP, forming a tightly bound, nucleotidefree
state while the second head is prevented from binding to the microtubule until
the nucleotide free, bound head binds ATP from solution. ATP-binding loosens the
neck-linker such that the second head can bind to the microtubule and release the
bound ADP [13–15]. When the hydrolysis product phosphate is released, the rear
head dissociates and the cycle starts again. Fluorescence microscopy allows us to
visualize the movement of single Kinesin-1 dimers under load-free conditions, and
optical trapping experiments make it possible to exert and measure forces.
3.3 Kinesins – structural adaptations for specific cellular tasks
Kinesins come in a variety of forms and functions, and not all are processive. We
also study non-processive kinesins which employ a myosin-like conformational change
of a lever-arm. One example is the dimeric Kinesin-14 ncd. This kinesin plays a
role in the formation of the meiotic and mitotic spindle. It is a minus-end-directed
motor, and kinetic and structural studies indicate that ncd dimers do not move
processively along a microtubule. We have detected the power stroke of ncd using
a double-laser trap and a so-called three bead assay. In the three bead assay, ncd
was adsorbed onto a large (5 µm) bead fixed to a coverslip. A microtubule was then
suspended and manipulated above the stationary bead with a dual-beam laser trap
(see Fig. 10, Ref. [17]). Figure 11 shows a typical time trace of the bead positions,
and the corresponding standard deviation from which binding events can be detected.
Because the data were noisy, the power stroke of about 9 nm could only be extracted
from ensemble-averaged events [16].
Kinesins differ not only in their processivity characteristics, but also in structure.
Some kinesins are monomeric and some are trimeric or tetrameric, adapted
for the specific roles in the cell. We currently investigate the motile behaviour of a