Disruption of the dynein–dynactin complex decreases the amount of Eg5 localizing to spindles in X. laevis egg extract. (a) Confocal fluorescence microscopy images of nonfixed spindles assembled either in the absence or additional presence of p50 or cc1 showing the localization of Eg5. The fluorescence of Eg5-paGFP (green) and of Cy5 microtubules (MT; red) was measured immediately after photoactivation of Eg5-paGFP in the entire spindle. (b) Fluorescence intensity profiles for photoactivated Eg5-paGFP and Cy5 microtubules along the spindle axis for the spindles shown in section a. (right) Mean ratios of the total fluorescence intensity of photoactivated Eg5-paGFP divided by the total fluorescence intensity of Cy5 microtubules (MT) for wild-type spindles, p50 spindles, and cc1 spindles are shown. The means were determined from five spindles per condition. The wild-type intensity ratio was set to 100. Error bars indicate standard deviation. (c) Confocal fluorescence microscopy images of fixed spindles assembled either in the absence (top) or presence (bottom) of cc1 showing the localization of dynein. Dynein heavy chain was detected by immunofluorescence (green), tubulin by using incorporated Alexa 568–tubulin (red), and DNA by Hoechst staining (blue). (d) Western blot analysis showing pull-down of p150^Glued with Eg5 on anti-Eg5 beads and pull-down of Eg5 with p150^Glued on anti-p150^Glued beads in X. laevis egg extract, either in the absence (−cc1) or presence (+cc1) of added p150 fragment cc1. Mock represents magnetic beads coated with an irrelevant antibody also incubated in X. laevis egg extract. An anti-p150^Glued antibody was used for detection in the top left three lanes and in the bottom right three lanes, whereas an anti-Eg5 antibody was used for detection in the top right three lanes and in the bottom left three lanes. All samples in a horizontal row were run on the same SDS gel. Molecular weight markers to the left of the blots are in kD.