Oscillations, Waves, and Interactions - GWDG

DPI60plus – a future with biophysics 451
Figure 14. Motility and inhibition of single and multiple Eg5Kin motors. (a) Displacement
and force produced by single truncated, C-terminally GFP-tagged Eg5Kin (Eg5Kin-GFP)
motors. Eg5Kin-GFP motors were sparsely covered on silica glass spheres and presented to
a microtubule using a single beam optical trap (g = 0.035 pN/nm). Eg5Kin-GFP moved
the bead processively out of the trap center producing an average force of 4.6 ± 0.1 pN.
Detachment occurs without observable stalling plateaus. (b) Kymograph of single Eg5Kin-
GFP, moving for micrometer-long distances along a TMR-labeled microtubule with an
average speed of 95 nm/s. Incremental, two-step bleaching was observed at points indicated
by arrows, quantitatively confirming the dimeric status of Eg5Kin (x-axis=325 s,
y-axis=11.32 µm). (c) Kymographs of single, GFP-tagged Eg5Kin motors moving along
microtubules at increasing Monastrol concentrations (x-axis=200 s, y-axis=10.53 µm). (d)
Graphical summary of motility data at increasing Monastrol concentrations: Eg5Kin-GFP
single molecule association time (red triangle down, IC50 = 6.5 µM), Eg5Kin-GFP single
molecule speeds (black triangles up), Eg5Kin multi motor surface gliding speeds (blue filled
squares). (From Lakämper et al., manuscript in preparation).
neck-linker of Eg5 attached to the stalk of Kinesin 1 moved in a highly processive
fashion along the microtubule for much longer distances than native Eg5. Monastrol
reduced the run-length, but neither the speed nor the binding frequency of single
dimeric chimeras (Fig. 14).
Fluorescence experiments are limited in two important ways. First, the number of
photons emitted by a single dye molecule on an individual motor protein is so small
that fast and accurate position detection is not possible. Second, one can neither