Figure 2.

SPDL-1 is recruited to the kinetochore by the RZZ complex. (A) SPDL-1 coimmunoprecipitates with Zwilch^ZWL-1. Worm extracts were depleted of Zwilch^ZWL-1 using an affinity-purified polyclonal antibody, and the resulting supernatant (S) and pellet (P) was analyzed by immunoblot. Loading of pellet is 20× relative to supernatant. An antibody against GFP was used in the control immunoprecipitation experiment. (B) SPDL-1 associates with the RZZ complex but is not a core subunit. A one-step immunoprecipitation of Zwilch^ZWL-1 and a stringent two-step isolation of GFP^LAP-tagged Zwilch^ZWL-1 were visualized on a silver-stained gel and analyzed by mass spectrometry as shown on the right. (C) Immunoblotting with the anti-Zwilch^ZWL-1 antibody detects a 70-kDa band, which is depleted >95% following RNAi. A cross-reacting protein band (*) serves as the loading control. (D) Immunofluorescence images of early embryos stained for SPDL-1 after depletion of Zwilch^ZWL-1 or ROD-1. Bars, 5 μm. (E) Depletion of SPDL-1 affects neither kinetochore targeting of RZZ subunits nor their rapid disappearance from kinetochores at anaphase onset. Selected frames from time-lapse sequences of embryos expressing GFP:Zwilch^ZWL-1, GFP:Zw10^CZW-1, and GFP:Spc24^KBP-4 are shown (see also Supplemental Movies 5, 6). Time is relative to the onset of sister chromatid separation. Bar, 5 μm.