Various Ways to Explore Processivity of Eg5 Motor Protein cd.

3. RESULTS AND DISCUSSION

3.1 Dependence of Microtubule Gliding Velocities on FL-Eg5 Concentration

For cross linking and 3-bead experiments it is necessary not only work with low concentrations of ATP molecules to slow down the motors, but also be close to single molecule level in the assay. For that performed experiments with different motor concentrations in order to obtain the low possible Eg5 dilution when the motility assay was still possible to observe. The Figure 3 shows the dependence of microtubule gliding velocities on FL-Eg5 concentration. This dependence is linear so the concentration of motor doesn’t change the average speed of microtubule sliding. The lowest Eg5 concentration obtained from this experiment is 0.05 mg/ml. When lowest concentrations of motor were used motility wasn't observed. Plot was fitted by Michaelis-Menten equation described in section 2.4.

Full-length Eg5 surface motility assays with different Eg5 concentrations were performed in the presence of methylcellulose. Methylcellulose is highly elongated polymer. Methylcellulose inhibits diffusion of actin filaments perpendicular (but not parallel) to their long axis; it thereby promotes motility at low motor densities by slowing the diffusion of the filament away from a surface during brief movements when all the motors are unbound [3]. From dependence of microtubule gliding velocity on FL-Eg5 concentration (see section 3.1) was obtained critical motor density. This dependence was shown on figure 2. Below 0.05 mg/ml FL-Eg5 concentration there was no microtubule attachment to the surface. By adding methylcellulose to the motility assay, microtubule gliding was observed below the critical Eg5 density and with 0.005 mg/ml Eg5 concentration motors heads on the surface still affect net microtubule sliding movement. In the presence of methylcellulose, the speed of microtubule of fixed length increases with the Eg5 density.