Directed movements of photoactivated Eg5-paGFP and of photobleached Cy5 microtubules along the spindle axis. (a) Spindles in egg extract depleted from endogenous Eg5 and supplemented with Eg5-paGFP and Cy5-tubulin before and after photoactivation of Eg5-paGFP and simultaneous photobleaching of Cy5 microtubules in two different spindle regions. Time series of confocal fluorescence microscopy images of Eg5-paGFP (green) and Cy5 microtubules (MT; red) at the indicated times, and fluorescence intensity profiles (Fig. 2) for Cy5 microtubules and Eg5-paGFP of the same spindle at the indicated times. (b) Intensity difference profiles for bleached Cy5 microtubules and intensity profiles for photoactivated Eg5-paGFP at the indicated times. Only parts of entire profiles are shown for the midzone, halfzone, and pole region. Intensity difference profiles for Cy5 microtubules appear inverted as compared with the intensity profiles (Fig. 2). Note the splitting of the initial single peak of the red microtubule profile in the midzone into two peaks of the blue microtubule profile after 56 s. A corresponding split is not observed in the Eg5 profiles. The intensity profile for the halfzone is derived from the spindle shown in section a, whereas the intensity profiles for the midzone and pole are derived from spindles not shown because of different imaging requirements for the different regions in the spindle. (c) Displacements of the peaks of photoactivated Eg5-paGFP (green) and of photobleached Cy5 microtubules (red) along the spindle axis with time. Linear regression fits (lines) to the experimental values (dots) yielded the velocities as indicated. (d, left) Box plots of the speeds of Eg5 movement (22 measurements in 19 spindles) and of microtubule flux (MT; 16 measurements). (right) Scatter diagram of the speeds of microtubule (MT) flux as a function of the speeds of Eg5 in the halfzone. Each data point represents two simultaneous measurements in the same spindle region.