FIGURE 2. PAR is required for bipolar spindle assembly and function.

a, PAR perturbation results in rapid loss of spindle structure. Cycled Xenopus egg extract spindles assembled in the presence of X-rhodamine-labelled tubulin were treated with 100 g ml^-1 PARG or 500 g ml^-1 purified anti-PAR antibody, and real-time images obtained every 30 s for 15 min on a spinning disk microscope. (See Supplementary Movie M1 (PARG addition) and Supplementary Movie M2 (PAR antibody addition) for full sequences.) b, PAR is required for spindle assembly and function. Addition of 100 g ml^-1 PARG for 2 h during (pre-assem.) and for 15 min after (15 min) spindle assembly both resulted in spindle monopoles with associated condensed chromosomes. These phenotypes are specific for PAR hydrolysis, as addition of 100 M ADP-HPD (+inhibitor), a PARG inhibitor, rescues spindle bipolarity. Tubulin is red and DNA is blue. c, Quantification of PAR hydrolysis. Equal amounts of CSF extract (lane 1) or cycled spindles reactions were untreated (lane 2), treated with 100 g ml^-1 PARG (lane 3), or 100 g ml^-1 PARG plus 100 M ADP-HPD (lane 4) and probed for antibodies against PAR (left blot) or tubulin (right blot). PARG treatment (lane 3) results in complete hydrolysis of PAR, whereas simultaneous addition of ADP-HPD partially protected PAR (lane 4). Molecular mass is indicated along the left (kDa). d, PAR is not required for microtubule aster structure or assembly. A total of 100 g ml^-1 PARG was added 25 min before DMSO- or RanGTP-induced (Ran) aster assembly in CSF extract, and real-time images obtained every 30 s for 15 min. PARG addition had no effect on aster assembly or structure. e, Eg5 and NuMA localization after PAR disruption. Cycled Xenopus extract spindles were not treated (top) or were treated with 100 g ml^-1 PARG (bottom), isolated and stained for tubulin (red) and Eg5 or NuMA (green), two proteins required for spindle assembly. Eg5 and NuMA remained associated with the resulting structures (PARG), suggesting that PAR may not be required for their targeting to spindles.